CA3173414A1 - Pd-1 agonist multimeric binding molecules - Google Patents

Abstract

This disclosure provides multimeric binding molecules that specifically and agonistically bind to programmed cell death protein 1 (PD-1). This disclosure also provides compositions comprising the multimeric binding molecules, polynucleotides that encode the multimeric binding molecules, and host cells that can produce the binding molecules. Further this disclosure provides methods of using the multimeric binding molecules, including methods for treating autoimmune disorders and preventing transplantation rejection.

Description

PD-I AGONIST MULTIMERIC BINDING MOLECULES
CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial Nos. 63/014,023, filed April 22, 2020; 63/050,413, filed July 10, 2020; and 63/144,708, filed February 2, 2021, which are each incorporated herein by reference in their entireties.
SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on April 20, 2021, is named 031W01-Sequence-Listing, and is 69,796 bytes in size.
BACKGROUND

Antibodies and antibody-like molecules that can multimeriz,e, such as IgA
and IgM
antibodies, have emerged as promising drug candidates, e.g., in the fields of immuno-oncology and infectious diseases, allowing for improved specificity, improved avidity, and the ability to bind to multiple binding targets. See, e.g., U.S. Patent Nos.
9,951,134, 9,938,347, 10,351,631, and 10,400,038, U.S. Patent Application Publication Nos. US
2019-0100597, US 2018-0009897, US 2019-0330374, US 2019-0330360, US 2019-0338040, US 2019-0338041, US 2019-0185570, US 2018-0265596, US 2018-0118816, US 2018-0118814, and US 2019-0002566, and PCT Publication Nos. WO 2018/187702, WO 2019/165340, and WO 2019/169314, the contents of which are incorporated herein by reference in their entireties.

Programmed cell death protein 1 (PD-1) is a cell surface receptor belonging to the immunoglobulin superfamily, which includes cell surface and soluble proteins that are involved with recognition, binding, and adhesion processes of cells. The initial members of this family were discovered due to their functional effect on augmenting T-cell proliferation following the addition of monoclonal antibodies (Hutloff et al.
(1999) Nature 397:263-266; Hansen et al. (1980) immunogenics 10:247-260). Two cell surface glyeoprotein ligands for PD-1, referred to as PD-Li and PD-L2, have been identified, and have been shown to downregulate T-cell activation and cytokine secretion upon binding to PD-.I (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8, Carter et al. (2002) Eur J Immunol 32:634-43; Obigashi et al.
(2005) Clin Cancer Res 11:2947-53). The PD-1 pathway has been implicated in a number of autoimmune diseases (Francisco et al., (2010) Inuriunol Rev 236: 219-42).
100051 There remains a need for therapeutics to treat autoimmune diseases, such as autoimmune diseases resulting from disruption of the PD-1 pathway.
SUMMARY

Provided herein is a multimeric binding molecule comprising two, five, or six bivalent binding units or variants or fragments thereof, where each binding unit comprises two IgA or IEM heavy chain constant regions or multimerizing fragments or variants thereof, each associated with a binding domain, where three to twelve of the binding domains are programmed cell death protein 1 (PD-1)-binding domains that specifically and agonistically bind to PD-1, where the binding molecule can activate PD-1-mediated signal transduction in a cell at a higher potency than an equivalent amount of a bivalent IgG antibody or fragment thereof comprising two of the same PD-I -binding domains, which also specifically binds to and agonizes PD-i. In some embodiments, the two, five, or six binding units are human, humanized, or chimeric immunoglobulin binding units.

In some embodiments, the three to twelve PD-I.-binding domains comprise a heavy chain variable region (VH) and a light chain variable region (VL), where the VII and VL
comprise six iinmunoglobulin complementarity determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where the HCDRI, HCDR2, HCDR3, LCDR I , LCDR2, and LCDR3 comprise the CDRs of an antibody comprising the VH and VL of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ Ill NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO:
25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ
ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or the VH of any one of SEQ ID
NO:
15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO:
24 and the VL of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ
ID NO: 22, or the CDRs of an antibody comprising the VII and VL of SEQ ID NO:
1 and

- 2 -SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: Sand SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ TD NO: 12, SEQ TD NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID
NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ
ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively with one or two single amino acid substitutions in one or more of the HCDRs or LCDRs, or the VH of any one of SEQ ID NO: 15, SEQ ID
NO:
17; SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VL
of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22 with .10 one or two single amino acid substitutions in one or more of the HCDRs or LCDRs.
[00081 In some embodiments, the three to twelve PD-1-binding domains comprise a heavy chain variable region (VH) and a light chain variable region (VL), where: (a) the VH and VL comprise six imm.unogiohulin complementarity determining regions HCDR.1, TICDR3, LCDR1, LCDR2, and LCDR3, where the LICDRI, TICDR2, TICDR3, LCDR1, LCDR2, and LCDR3 comprise the CDRs of an antibody comprising the VH and VI, of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID
NO:
5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO:

10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID
NO: 25 and SEQ ID NO: 26, SEQ Ill NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO:
34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively with zero, one, or two single amino acid substitutions in one or more of the HCDRs or LCDRs; (b) the VH and VL
comprise six immunoglobulin complementarity determining regions HCDR.1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where the HCDR1, HCDR2, HCDR3. LCDR1, LCDR2, and LCDR3 comprise the CDRs of an antibody comprising the VH of any one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ 113 NO: 21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VI, of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID
NO:
20, or SEQ ID NO: 22 with zero, one, or two single amino acid substitutions in one or more of the HCDRs or LCDRs; (c) the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO:
1 and SEQ
ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID

NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ
ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ED NO: 26,

- 3 -SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO:
31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ
ID NO: 50, respectively; or (d) the VH comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to of any one of SEQ
ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID
NO: 20, or SEQ ID NO: 22.
100091 In some embodiments, the VH and VI, comprise six immunoglobulin .10 complementarity determining regions FICDR1, T-ICDR2, FICDR3, LCDRI, LCDR2, and I ,CDR3, where the HCDR1, HCDR2, HCDR3, T,CDRI, TEDR2, and I,CDR3 comprise the CDRs of an antibody comprising the VH and VL of SEQ ID NO: 1 and SEQ ID
NO:
2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 13 and SEQ ID NO: 14, and SEQ ID
NO: 25 and SEQ ID NO: 26, respectively, or the CDIts of an antibody comprising the VII
and VL of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID
NO: 13 and SEQ ID NO: 14, and SEQ ID NO: 25 and SEQ ID NO: 26, respectively with one or two single amino acid substitutions in one or more of the LICDRs or LCDRs.
(00101 In some embodiments, the three to twelve PD-1-binding domains of the binding molecule comprise an antibody VH and a VL, where the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100%
identical to SEQ
ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ

ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ
ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ TD NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID
NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or the VH of any one of SEQ ID
NO:
15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO:
24 and the VL of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ
ID NO: 22. In some embodiments, the three to twelve PD-1-binding domains of the binding molecule comprise an antibody VH and a VIõ where the VH and VI, comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100%
identical to SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ

ID NO: 13 and SEQ ID NO: 14, and SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

- 4 -100111 In some embodiments, the three to twelve PD-1-binding domains comprise antibody VH and VI, regions comprising the amino acid sequences SEQ ID NO: 1 and SEQ ID
NO:
2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO:
12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID
NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID
NO: 50, respectively, or the VH of any one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID
NO: 19, SEQ ID NO: 21., SEQ ID NO: 23, or SEQ ID NO: 24 and the VL of any one of .10 SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22. In some embodiments, the three to twelve PD-1-binding domains comprise antibody VH and VI.
regions comprising the amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2, SEQ
ID
NO: 3 and SEQ ID NO: 4, SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO: 25 and SEQ ID NO: 26, respectively.
100121 In some embodiments, each binding unit comprises two heavy chains and two light chains, where the heavy chains and light chains comprise VH and VL amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100%
identical to SEQ
ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ

ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ Ill NO: 10, SEQ
ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ TD
NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or the VII of any one of SEQ ID
NO:
15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO:
24 and the 'VL of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ
ID NO: 22. In some embodiments, each binding unit comprises two heavy chains and two light chains, where the heavy chains and light chains comprise VH and VL amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100%
identical to SEQ
ID NO: I and SEQ ID NO: 2, SEQ Ill NO: 3 and SEQ ID NO: 4, SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO: 25 and SEQ ID NO: 26, respectively.
100131 In some embodiments, the heavy chains and light chains comprise the VH
and VL
amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID
NO:
4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9

- 5 -

6 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID
NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ
ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or the VH
of any one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID
NO: 23, or SEQ ID NO: 24 and the VL of any one of SEQ ID NO: 16, SEQ ID NO:
18, SEQ ID NO: 20, or SEQ ID NO: 22. In some embodiments, the heavy chains and light chains comprise the VH and VL amino acid sequences SEQ ID NO: 1 and SEQ ID NO:
2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO:
.10 25 and SEQ ID NO: 26, respectively.
[00141 In some embodiments, the multimeric binding molecule is a. dimeric binding molecule comprising two bivalent IgA or IgA-like binding units and a J chain or functional fragment or variant thereof, where each binding unit comprises two IgA heavy chain constant regions or multimerizing fragments or variants thereof, each comprising an IgA
Ca3 domain and an IgA tailpiece domain. In some embodiments, each IgA heavy chain constant region or multimerizing fragment or variant thereof further comprises a Cal domain, a Ca2 domain, an IgA hinge region, or any combination thereof. In some embodiments, the IgA heavy chain constant regions or multimerizing fragments thereof arc human IgA constant regions. In some embodiments, each binding unit comprises two IgA heavy chains each comprising a VH situated amino terminal to the IgA
constant region or multimerizing fragment thereof, and two immunoglobulin light chains each comprising a 'VL situated amino terminal to an immunoglobulin light chain constant region.

In some embodiments, the multimeric binding molecule is a pentaineric or a hexameric binding molecule comprising five or six bivalent IgM binding units, respectively, where each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments thereof each associated with a PD-1-binding domain, where each IgM heavy chain constant region. comprises an IgM CO and IgM tailpiece domain.
In some embodiments, the IgM heavy chain constant regions or fragments or variants thereof each further comprise a Cul domain, a Cj,i2 domain, a CP domain, or any combination thereof. In some embodiments, the IgM heavy chain constant region is a human IgM
constant region.
[00161 In some embodiments, each binding unit comprises two IgM heavy chains each comprising a VH situated amino terminal to the IgM constant region or fragment thereof, and tvvo immunoglobulin light chains each comprising a VL situated amino terminal to an irnmunoglobulin light chain constant region.
[00171 In some embodiments, the multimeric binding molecule comprises SEQ ID
NO: 35, SEQ ID NO: 36, or a multimerizing fragment thereof. In some embodiments, the IgM
constant region comprises a substitution relative to a wild-type human IgM
constant region at position 310, 311, 313, and/or 315 of SEQ ID NO: 35 or SEQ TD NO: 36. In some embodiments, the IgM constant region comprises two or more substitutions relative to a wild-type human IgM constant region at positions 46,209, 272, or 440 of SEQ ID
NO: 35 or SEQ ID NO: 36.
.10 [00181 In some embodiments, the multimeric binding molecule is pentameric, and further comprises a .1-chain or functional fragment or variant thereof.
[00191 In some embodiments, the J-chain or functional fragment or variant thereof is a variant J-chain comprising one or more single amino acid substitutions, deletions, or insertions relative to a wild-type J-chain that can affect serum half-life of the multimeric binding molecule; and where the multimeric binding molecule comprising the variant .1f-chain exhibits an. increased serum half-life upon administration to an animal relative to a reference multimeric binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions, and is administered in the same way to the samc animal species.
100201 In some embodiments, the J-chain or functional fragment thereof comprises an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human j-chain (SEQ ID NO: 41). In some embodiments, the amino acid corresponding to Y102 of SEQ ID NO: 41 is substituted with alanine (A), scrim (S), or arginine (R). In some embodiments, the amino acid corresponding to Y102 of SEQ
NO: 41 is substituted with alanine (A).

In some embodiments, the J-chain is a variant human J-chain and comprises the amino acid sequence SEQ ID NO: 42. In some embodiments, the J-chain or functional fragment thereof comprises an amino acid substitution at the amino acid position corresponding to amino acid N49, amino acid S51, or both N49 and S51 of the mature human J-chain (SEQ ID NO: 41), where a single amino acid substitution corresponding to position S51 of SEQ ID NO: 41 is not a threonine (T) substitution. In some embodiments, the position corresponding to N49 of SEQ ID NO: 41 is substituted with alanine (A), glycine (G), threonine 0), senile (S) or aspartic acid (D). In some embodiments, the

- 7 -position corresponding to N49 of SEQ ID NO: 41 or SEQ ID NO: 42 is substituted with alanine (A). in some embodiments, the position corresponding to S51 of SEQ H.) NO: 41 or SEQ ID NO: 42 is substituted with alanine (A) or glycine (0). In some embodiments, the position corresponding to S51 of SEQ ID NO: 41 or SEQ ID NO: 42 is substituted with alanine (A.).

In some embodiments, the J-chain or functional fragment or variant thereof further comprises a heterologous polypeptide, where the heterologous polypeptide is directly or indirectly fused to the .1-chain or functional fragment or variant thereof.

In some embodiments, the heterologous polypeptide is fused to the J-chain or fragment thereof via a peptide linker. In some embodiments, the peptide linker comprises at least 5 amino acids, but no more than 25 amino acids. In some embodiments, the J-chain or functional fragment or variant thereof further comprises a heterologous polypeptide, where the heterologous polypeptide is fused to the J-chain or functional fragment or variant thereof via a peptide linker comprising at least 5 amino acids, but no more than 25 amino acids. In some embodiments, the peptide linker consists of GGGGS (SEQ ID
NO:
43), 000GS0000S (SEQ ID NO: 44), GGGGSGGGGSGGGGS (SEQ ID NO: 45), GGGGSGGGGSGGGGSGCiGGS (SEQ ID NO: 46), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 47). In some embodiments, the heterologous polypeptidc is fused to the N-terminus of the J-chain or fragment or variant thereof, the C-terminus of the J-chain or fragment or variant thereof, or to both the N-term inus and C-terminus of the J-chain or fragment or variant thereof.

In some embodiments, the heterologous polypeptide can influence the absorption, distribution, metabolism and/or excretion (AI)ME) of the multirneric binding molecule.

In some embodiments, the heterologous polypeptide comprises an antigen binding domain. In some embodiments, the antigen binding domain of the heterologous polypeptide is an antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment comprises an Fab fragment, an Fab' fragment, an F(a13')2 fragment, an H fragment, an Fv fragment, a single-chain Fv (scFv) fragment, a disulfide-linked 17v (sdFv) fragment, or any combination thereof. In some embodiments, the antigen-binding fragment is a scFv fragment. In some embodiments, the antigen binding domain binds ICOS Ligand (ICOSLG), ICOS (CD278), Interleuldn 6 (IL6), CD28, CD3, CD80, CD86, Tumor Necrosis Factor Alpha (TNFa), or Fibroblast Activation Protein (FAP).

- 8 -Also provided herein is a composition comprising the multimeric binding molecule disclosed herein. In some embodiments, the composition further comprises a pharmacologically acceptable excipient.

Also provided herein is a polynucleotide comprising a nucleic acid sequence that encodes a polypeptide subunit of the binding molecule disclosed herein. In some embodiments, the poly-peptide subunit comprises an IgM heavy chain constant region and at least an antibody VH portion of the PD-1-binding domain of the multimeric binding molecule.
100281 In some embodiments, the polypeptide subunit comprises a human IgM
constant .10 region or fragment thereof fused to the CAerminal end of a VH comprising: (a) I{CDR1, HCDR2., and HCDR3 regions comprising the CDRs contained in the V-14 amino acid sequences SEQ ID NO: I, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:

9, SEQ ID NO: 1 I., SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ
ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID
NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 49, or the CDRs contained in the VH amino acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11., SEQ ID NO: 13, SEQ TD NO: 15, SEQ ID NO:
17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ Ill NO: 33, or SEQ ID NO: 49 with one or two single amino acid substitutions in one or more of the HCDR.s;
or (b) an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100%
identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID
NO:
9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ
ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 49.

In some embodiments, the polypeptide subunit comprises a light chain constant region and an antibody VI. portion of the PD-1.-binding domain of the multimeric binding molecule. hi some embodiments, the polypeptide subunit comprises a human kappa or lambda light chain constant region or fragment thereof fused to the C-terminal end of a VL comprising: (a) LCDR.1, LCDR2, and LCDR3 regions comprising the CDRs contained in the VL amino acid sequences SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ
ID
NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:
18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 50, or the CDRs contained in the VL
amino acid sequences SEQ ID NO: 2, SEQ ID NO: 4, SEQ TT) NO: 6, SEQ ID NO: 8, SEQ
ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 1.8, SEQ ID

NO: 20, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:
32, SEQ ID NO: 34, or SEQ ID NO: 50 with one or two single amino acid substitutions in one or more of the LCDRs; or (b) an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ
ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: :20. SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ
.10 ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 50.
[00301 Also provided herein is a composition comprising a polynucleotide provided herein.
In some embodiments, the composition comprises a polynucleotide comprising a nucleic acid sequence that encodes an IgM heavy chain constant region and at least an antibody VII portion of the PD-1-binding domain of a multimeric binding molecule provided herein, and a poly-nucleotide comprising a nucleic acid sequence that encodes a light chain constant region and an. antibody VI, portion of the PD-1.-binding domain of a multimeric binding molecule provided herein. In some embodiments, the polynucleotides are on separate vectors. In some embodiments, the polynucleotides are on a single vector. In some embodiments, the composition further comprises a polynucleotide comprising a nucleic acid sequence encoding a J chain, or a functional fragment thereof, or a functional variant thereof.
100311 Also provided herein is a vector or vectors disclosed herein.

Also provided herein is a host cell comprising a polynucleotide provided herein, a composition provided herein, or a vector or vectors provided herein, where the host cell can express a binding molecule provided herein, or a subunit thereof.
100331 Also provided herein is a method of producing a binding molecule provided herein, comprising culturing a host cell provided herein, and recovering the binding molecule.

Also provided herein is a method for treating an autobrunnine disorder, an inflammatory disorder, or a combination thereof in a subject in need of treatment comprising administering to the subject an effective amount of a multimeric binding molecule provided herein, where the multimeric binding molecule exhibits greater potency than an equivalent amount of a monomeric or dimeric binding molecule binding to the same binding partner. In some embodiments, the subject is human.

- 10 -Also provided herein is a method for preventing transplantation rejection in a subject, comprising administering to the subject an effective amount of a multimeric binding molecule provided herein, where the multimeric binding molecule exhibits greater potency than an equivalent amount of a monomeric or dimeric binding molecule binding to the same binding partner, and where the subject is a transplantation recipient. In some embodiments, the subject is human.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. I shows binding of anti-PD-I. IgG #1, anti-PD-I IgM #1. anti-PD-I IgG
#2, and anti-PD-1 IgM #2 to human PD-I in an ELISA assay.
(00371 FIG. 2 shows binding of anti-PD-1 IgG #1, anti-PD-1 IgM #1, anti-PD-I IgG #2, and anti-PD-1 IgM #2 to human PD-I in a cell-based assay.

FIGS. 3A-3B show activation of PD-I signaling by anti-PD-1 IgG or IgM #1 (FIG.
3A) or anti-PD-I IgG or IgM #2 (FIG. 3B).
100391 FIGS. 4A-4B show activation of PD-1 signaling by anti-PD-1 IgG, IgG +
cross-linker, or IgM #1 (FIG. 4A) or anti-PD-I IgG, IgG + cross-linker, or IgM #2 (FIG. 4B).

FIGS. 5A-5D show activation of PD-1 signaling by anti-PD-1. IgG, IgG +
cross-linker, or IgM 41 (FIG. 5A) or anti-PD-1 IgG, IgG + cross-linker, or IgM #2 (FIG. 5B) or anti-PD-1 IgG, IgG + cross-linker, or IgM #3 (FIG. 5C) or anti-PD-1 IgG, 1gG +
cross-linker, or IgM #4 (FIG. 5D).
100411 FIGS. 6A-6D
show activation of PD-1 signaling by anti-PD-1 IgG, IgG + cross-linker, pentameric IgM, or hexarneric IgHM #1 (FIG. 6A) or anti-PD-I IgG, IgG
+ cross-linker, pentameric IgM, or hexameric IgHM #2 (FIG. 6B) or anti-PD-I IgG, IgG +
cross-linker, pentameric IgM, or hexarneric IgHM #3 (FIG. 6C) or anti-PD-1 IgG, IgG
+ cross-linker, pentameric IgM., or hexameric IgHM 45 (FIG. 6D).
DETAILED DESCRIPTION
Definitions As used herein, the term "a" or "an" entity refers to one or more of that entity; for example, "a binding molecule," is understood to represent one or more binding molecules.
As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

- 11 -Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B,"
"A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, 13, or C; A or C; A or B; B or C; A and C; A and B; B and C; A
(alone); B
(alone); and C (alone).

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this .10 disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, J110, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.
100451 Units, prefixes, and symbols are denoted in their Systeme International de Unites (Si) accepted form. Numeric ranges axe inclusive of the numbers defining the range.
Unless otherwise indicated, amino acid sequences are written left to right in amino to carbox.y orientation. The headings provided herein are not limitations of the various embodiments or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "poly-peptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain.," or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of any of these terms. The term "polypeptide"
is also intended to refer to the products of post-expression modifications of the poly-peptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, and derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived

- 12 -from a biological source or produced by recombinant technology but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
100471 A polypeptide as disclosed herein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such structure.
Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can .10 adopt many different conformations and are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of an amino acid, e.g., a serine or an asparagine. Asparagine (N)-linked glycans are described in more detail elsewhere in this disclosure.
100481 By an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required.
For example, an isolated polypeptide can be removed from its native or natural environment. Recombinandy produced polypeptides and proteins expressed in host cells arc considered isolated as disclosed herein, as arc native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
100491 As used herein, the term -a non-naturally occurring polypeptide" or any grammatical variants thereof, is a conditional definition that explicitly excludes, but only excludes, those forms of the polypeptide that are, or might be, determined or interpreted by a judge or an administrative or judicial body, to be "nan.trally-occurring."

Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms "fragment,"
"variant," "derivative" and "analog" as disclosed herein include any polypeptides which retain at least some of the properties of the corresponding native antibody or polypeptide, for example, specifically binding to an antigen. Fragments of poly-peptides include, for example, proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of, e.g., a polypeptide include fragments as described above, and also polypeptides with altered amino acid sequences

- 13 -due to amino acid substitutions, deletions, or insertions. In certain embodiments, variants can be non-naturally occurring.. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions, or additions.
Derivatives are polypeptides that have been altered so as to exhibit additional features not found on the original poly-peptide. Examples include fusion proteins. As used herein a "derivative" of a polypeptide can also refer to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those polypeptides that contain one or more derivatives of the twenty .10 standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistirline can be substituted for histidine; homoserine can be substituted for serine; and omithine can be substituted for lysine.
10051.1 A "conservative amino acid substitution" is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,. glycineõ alaninc, valinc, lcucinc, isolcucinc, prolinc, phenylalanineõ
methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
For example, substitution of a phenylalanine for a tyrosine is a conservative substitution.
In certain embodiments, conservative substitutions in the sequences of the polypeptides, binding molecules, and antibodies of the present disclosure do not abrogate the binding of the polypeptide, binding molecule, or antibody containing the amino acid sequence, to the antigen to which the antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen-binding are well-known in the art (see, e.g., Bn]mmell et al. Biochem . 32: 1180-1 187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA
94:.412-417 (1997)).

The term "polynucleotid.e" is intended to encompass a singular nucleic acid as well as plural nucleic acids and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (m RNA), cDNA, or plasmid DNA (pDNA). A polynucleotide can

- 14 -comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The terms "nucleic acid"
or "nucleic acid sequence" refer to any one or more nucleic acid segments, e.g., DNA or RNA
fragments, present in a polynucleotide.
100531 By an "isolated" nucleic acid or polynucleotide is intended any form of the nucleic acid or polynucleotide that is separated from its native environment. For example, gel-purified polynucleotide, or a recombinant polynucleotide encoding a polypeptide contained in a vector would be considered to be "isolated." Also, a polynucleotide segment, e.g., a PCR product, which has been engineered to have restriction sites for .10 cloning is considered to be "isolated." Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in a non-native solution such as a buffer or saline. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides, where the transcript is not one that would be found in nature. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. in addition, poly-nucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

As used herein, the term "a non-naturally occurring polynucleotide" or any grammatical variants thereof, is a conditional dcfmition that explicitly excludes, but only excludes, those forms of the nucleic acid or polynucleotide that are, or might be., determined or interpreted by a judge, or an administrative or judicial body, to be "naturally-occurring."

As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA. or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors.
Furthermore, any vector can contain a single coding region, or can comprise two or more coding regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid can include lieterologous coding regions, either fused or unfused to another

- 15 -coding region. Heterologous coding regions include without limitation, those encoding specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
[00561 In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in. such a way as to place expression of the gene product under the influence or .10 control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
The promoter can be a cell-specific promoter that directs substantial transcription of the DNA in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.

A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit a-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lym.phokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
[00581 Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation

- 16 -initiation and termination codons, and elements derived from picomaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE
sequence).
100591 In other embodiments, a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

Polynucleotide and nucleic acid coding regions can be associated with additional.
coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein .10 chain across the rough endoplasmic reticulurn has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells can have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide.
In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mousc13-glucuronidase.
10061.1 As used herein, the term 'binding molecule" refers in its broadest sense to a molecule that specifically binds to a receptor or target, e.g., an epitope or an antigenic determinant. As described further herein, a binding molecule can comprise one of more "binding domains," e.g., "antigen-binding domains" described herein. A non-limiting example of a binding molecule is an antibody or antibody-like molecule as described in detail herein that retains antigen-specific binding. In certain embodiments a "binding molecule" comprises an antibody or antibody-like or antibody-derived molecule as described in detail herein.

As used herein, the terms "binding domain" or "antigen-binding domain" (can be used interchangeably) refer to a region of a binding molecule, e.g., an antibody or antibody-like, or antibody-derived molecule, that is necessary and sufficient to specifically bind to a target, e.g., an epitope, a polypeptide, a cell, or an organ. For example, an e.g., a heavy chain variable region and a light chain variable region of an antibody, either as two separate polypeptide subunits or as a single chain, is considered to be a "binding

- 17 -domain." Other antigen-binding domains include, without limitation, a single domain heavy chain variable region (VHH) of an antibody derived from a camelid species, or six immunoglobulin complementarity determining regions (CDRs) expressed in a fibronectin scaffold. A "binding molecule," e.g., an "antibody" as described herein can include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more "antigen-binding domains."
100631 The team "antibody" and "iinnumoglobulin" can be used interchangeably herein.
An antibody (or a fragment, variant, or derivative thereof as disclosed herein, e.g., an IgM-like antibody) includes at least the variable domain of a heav-y chain (e.g., from a camelid .10 species) or at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). Unless otherwise stated, the term "antibody" encompasses anything ranging from a small antigen-binding fragment of an antibody to a full sized antibody, e.g., an IgG
antibody that includes two complete heavy chains and two complete light chains, an IgA
antibody that includes four complete heavy chains and four complete light chains and includes a J-chain and/or a secretory component, or an IgM-derived binding molecule, e.g., an IgM antibody or IgM-like antibody, that includes ten or twelve complete heavy chains and ten or twelve complete light chains and optionally includes a J-chain or functional fragment or variant thereof.
100641 The term "immunoglobulin" comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (7, IA, Ct, 8, s) with some subclasses among them (e.g., y.1-74 or al-a2)). It is the nature of this chain that determines 75 the "isotype" of the antibody as IgG, IgM, IgA IgD, or IgE, respectively. The immunoglobulin subclasses (subtypes) e.g., IgG I., IgG2, igG3, IgG4, TgA 1, IgA2, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these immunoglobulins are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within, the scope of this disclosure.
[0065] Light chains are classified as either kappa or lambda (K, A.). Each heavy chain class can be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the

- 18 -immunoglobulins are expressed, e.g., by hybridomas. B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. The basic structure of certain antibodies, e.g., IgG antibodies, includes two heavy chain subunits and two light chain subunits covalently connected via disulfide bonds to form a "Y" structure, also referred to herein as an "1-12L2" structure, or a "binding unit."

The term "binding unit" is used herein to refer to the portion of a binding molecule, e.g., an antibody, antibody-like molecule, or antibody-derived molecule, antigen-binding fragment thereof, or multimerizing fragment thereof, which corresponds to a standard .10 "ii2L2" immunoglobulin structure, i.e., two heavy chains or fragments thereof and two light chains or fragments thereof. In certain embodiments, e.g., where the binding molecule is a bivalent IgG antibody or antigen-binding fragment thereof, the terms "binding molecule" and "binding unit" are equivalent. In other embodiments, e.g., where the binding molecule is a "multimeric binding molecule," e.g., a dimeric IgA
antibody, a dimeric IgA-like antibody, a dimeric IgA-derived binding molecule, a pentameric or hexameric IgM antibody, a pentameric or hexameric IgM-like antibody, or a pentain.eric or hexameric IgM-derived binding molecule or any derivative thereof, the binding molecule comprises two or more "binding units." Two in the case of an IgA
dimer, or five or six in the case of an IgM pcntamer or hexamer, respectively. A binding unit need not include MI-length antibody heavy and light chains, but will typically be bivalent; i.e., will include two "antigen-binding domains," as defined above. As used herein, certain binding molecules provided in this disclosure are "dimeric," and include two bivalent binding units that include IgA constant regions or multimerizing fragments thereof Certain binding molecules provided in this disclosure are "pentarneric" or "hexameric," and include five or six bivalent binding units that include IgM constant regions or multimerizing fragments or variants thereof. A binding molecule, e.g., an antibody or antibody-like molecule or antibody-derived binding molecule, comprising two or more, e.g., two, five, or six binding units, is referred to herein as "inultimeric."
100671 The term "J-chain" as used herein refers to the J-chain of IgM or IgA
antibodies of any animal species, any functional fragment thereof, derivative thereof, ancUor variant thereof, including a mature human J-chain, the amino acid sequence of which is presented as SEQ ID NO: 41. Various J-chain variants and modified J-chain derivatives are disclosed herein. As persons of ordinary skill in the art will recognize, "a functional fragment" or "a

- 19 -functional variant" includes those fragments and variants that can associate with IgM
heavy chain constant regions to fonri a pentameric IgM antibody.
[00681 The term "modified J-chain- is used herein to refer to a derivative of a J-chain polypeptide comprising a heterologous moiety, e.g., a heterologous polypeptide, e.g., an extraneous binding domain or functional domain introduced into or attached to the J-chain sequence. The introduction can be achieved by any means, including direct or indirect fusion of the heterologous polypeptide or other moiety or by attachment through a peptide or chemical linker. The term "modified human J-chain- encompasses, without limitation, a native sequence human J-chain comprising the amino acid sequence of SEQ ID
NO: 41 .10 or functional fragment thereof, or functional variant thereof, modified by the introduction of a heterologous moiety, e.g., a heterologous polypeptide, e.g., an extraneous binding domain. In certain embodiments the heterologous moiety does not interfere with efficient polymerization of IgM into a pentamer or IgA into a dirtier, and binding of such polymers to a target. Exemplary modified J-chains can be found, e.g., in U.S. Patent Nos. 9,951,134 and 10,400,038, and in U.S. Patent Application Publication Nos. US-2019-0185570 and US-2018-0265596, each of which is incorporated herein by reference in its entirety.
[00691 As used herein the term "IgM-derived binding molecule" refers collectively to native IgM antibodies, IgM-like antibodies, as well as other IgM-derived binding molecules comprising non-antibody binding and/or functional domains instead of an antibody antigen binding domain or subunit thereof, and any fragments, e.g., multimerizing fragments, variants, or derivatives thereof.

As used herein, the term "IgM-like antibody" refers generally to a variant antibody or antibody-derived binding molecule that still retains the ability to form hemmers or pentamers, e.g., in association with a J-chain. An IgM-like antibody or other IgM-derived binding molecule typically includes at least the Cp.4-tp domains of the IgM
constant region but can include heavy chain constant region domains from other antibody isotypes, e.g., IgG, from the same species or from a different species. An IgM-like antibody or other IgM-derived binding molecule can likewise be an antibody fragment in which one or more constant regions are deleted, as long as the IgM-like antibody is capable of forming hexamers and/or pentamers. Thus, an IgM-like antibody or other IgM-derived binding molecule can be, e.g., a hybrid IgM/IgG antibody or can be a "multimerizing fragment" of an IgM antibody.

- 20 -The terms "valency," "bivalent," "multivalent" and grammatical equivalents, refer to the number of binding domains, e.g., antigen-binding domains in given binding molecule, e.g., antibody, antibody-derived, or antibody-like molecule, or in a given binding unit. As such, the terms "bivalent", "tetravalent", and "hexavalent"
in reference to a given binding molecule, e.g., an IgM antibody, IgM-like antibody, other IgM-derived binding molecule, or multimerizing fragment thereof, denote the presence of two antigen-binding domains, four antigen-binding domains, and six antigen-binding domains, respectively. A typical IgM antibody, IgM-like antibody, or other 1gM-derived binding molecule, where each binding unit is bivalent, can have 10 or 12 valencies. A
bivalent or .10 multivalent binding molecule, e.g., antibody or antibody-derived molecule, can be rnonospecific, .e., all of the antigen-binding domains are the same, or can be bispecific or multispecific, e.g., where two or more antigen-binding domains are different, e.g., bind to different epitopes on the same antigen, or bind to entirely different antigens.

The term "epitope" includes any molecular determinant capable of specific binding to an antigen-binding domain of an antibody, antibody-like, or antibody-derived molecule.
In certain embodiments, an. epitope can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, can have three-dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of a target that is bound by an antigen-binding domain of an antibody.

The term "target" is used in the broadest sense to include substances that can be bound by a binding molecule, e.g., antibody, antibody-like, or antibody-derived molecule.
A. target can be, e.g., a polypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule, or a minimal epitope on such molecule. Moreover, a "target" can, for example, be a cell, an organ, or an organism, e.g., an animal, plant, microbe, or virus, that comprises an epitope that can be bound by a binding molecule, e.g., antibody, antibody-like, or antibody-derived molecule.

Both the light and heavy chains of antibodies, antibody-like, or antibody-derived molecules are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the variable light (VL) and variable heavy (VI-1) chain portions determine antigen recognition and specificity. Conversely, the constant region domains of the light chain (CL) and the heavy chain (e.g., CHI , Cl-12. CH3, or CH4) confer

- 21 -biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N-tenninal portion is a variable region and at the C-terminal portion is a constant region; the CH3 (or CH4, e.g., in the case of leM) and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.
[0075]
A "full length IgM antibody heavy chain" is a polypeptide that includes, in N-terminal to C-tenninal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CM I or Cgl), an antibody heavy chain constant .10 domain 2 (CM2 or Cg2), an antibody heavy chain constant domain 3 (CM3 or Cg3), and an antibody heavy chain constant domain 4 (CM4 or Cp4) that can include a.
tailpiece.

As indicated above, variable region(s) allow a binding molecule, e.g., antibody, antibody-like, or antibody-derived molecule, to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VII domain, or subset of the complementarity determining regions (Calks), of a binding molecule, e.g., an antibody, antibody-like, or antibody-derived molecule, combine to form the antigen-binding domain. More specifically, an antigen-binding domain can be defined by three CDRs on each of the VH and VL chains. Certain antibodies form larger structures. For example, IgA can form a molecule that includes two H2L2 binding units and a J-chain covalently connected via disulfide bonds, which can be further associated with a secretory component, and IgM can form a pentameric or hexarneric molecule that includes five or six H2L2 binding units and optionally a j-chain covalently connected via disulfide bonds.
[0077] The six "complementarity determining regions" or "CDRs" present in an antibody antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domain, referred to as "framework" regions, show less inter-molecular variability. The framework regions largely adopt a 13-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the 13-sheet structure.
Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding

-22 -of the antibody to its cognate epitope. The amino acids that make up the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been defined in various different ways (see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J.
Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their entireties).

In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all .10 such meanings unless explicitly stated to the contrary. A specific example is the use of the term "cornplementarity determining region" ("CDR") to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described, for example, by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference The Kabat and Chothia definitions include overlapping or subsets of amino acids when compared against each other. Nevertheless, application of either definition (or other definitions known to those of ordinary skill in the art) to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein, unless otherwise indicated. The appropriate amino acids which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. The exact amino acid numbers which encompass a particular CDR. will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which amino acids comprise a particular CDR given the variable region amino acid sequence of the antibody.
Table 1 CDR. Definitions*
Kabat Chothia VL CD1t3 89-97 91-96

- 23 -*Numbering of all CDR definitions in Table 1 is according to the numbering conventions set forth by Kabat et at. (see below).
100791 Antibody variable domains can also be analyzed, e.g., using the IMGT
information system (imgt_dot_cines_dot_fri) (IMGTON-Quest) to identify variable region segments, including CDRs. (See, e.g., Brochet et al., Nucl. Acids Res. 36:W503-508, 2008).

Kabat et at. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by 'Cabal et at., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless use of the Kabat numbering system is explicitly noted, however, consecutive numbering is used for all amino acid sequences in this disclosure.
100811 The Kabat numbering system for the human IgM constant domain can be found in Kabat, et. al. "Tabulation and Analysis of Amino acid and nucleic acid Sequences of Precursors, V-Regions, C-Regions, J-Chain, T-Cell Receptors for Antigen, T-Cell Surface Antigens, 0-2 Microglobulins, Major Histocompatibility Antigens, Thy-1, Complement, C-Reactive Protein, Thymopoictin, Integrins, Post-gamma. Globulin, a-2 Macroglobulins, and Other Related Proteins," U.S. Dept. of Health and Human Services (1991).
IgM
constant regions can be numbered sequentially (i.e., amino acid 141 starting with the first amino acid of the constant region, or by usin.g the Kabat numbering scheme. A
comparison of the numbering of two alleles of the human IgM constant region sequentially (presented herein as SEQ ID NO: 35 (allele 1GHM*03) and SEQ ID NO: 36 (allele IGHM*04)) and by the Kabat system is set out below. The underlined amino acid residues are not accounted for in the Kabat system ("X," double underlined below, can be serine (S) (SEQ
ID NO:
35) or glycine (G) (SEQ ID NO: 36)):
Sequential (SEQ ID NO: 35 or SEQ ID NO: 36)/KABAT numbering key for IgM heavy chain

- 24 -(00821 Binding molecules, e.g., antibodies, antibody-like, or antibody-derived molecules, antigen-binding fragments, variants, or derivatives thereof, and/or multimerizing fragments thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chitneric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab. Fab' and F(a131)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VII domain, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.
[00831 By "specifically binds," it is generally meant that a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain, and the epitope. According to this definition, a binding molecule, e.g., antibody, antibody-like, or antibody-derived molecule, is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify' the relative affinity by which a certain binding molecule binds to a certain epitope. For example, binding molecule "A" can be deemed to have a higher specificity for a given epitope than binding molecule "B," or binding molecule "A" can be said to bind to cpitopc "C" with a higher specificity than. it has for related epitope "D."

A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof disclosed herein can be said to bind a target antigen with an off rate (k(off)) of less than or equal to 5 X 10-2 sec-1, 10-2 sec-1, 5 X 10-3 sec-1, 10-3 sec-1, 5 X 10-4 sec-1, 10-4 sec-1,5 X 10-5 sec-1., or 10-5 sec-i5 X 10-6 sec-I, 10-6 sec-1, 5 X 10-7 sec-I or 10-7 sec-1.
[00851 A binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative disclosed herein can be said to bind a target antigen with an on rate (k(on)) of

-25 -greater than or equal to 103 M-1 sec-1, 5 X 103 M-1 sec-1, 104 M-1 sec-1, 5 X

sec-1. 105 M-1 sec-I , 5 X 105 M-I sec-1, 106 M-1 sec-1. or 5 X 106 M-1 sec-1 or 107 M-1 sec-1.
[00861 A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof is said to competitively inhibit binding of a reference antibody or antigen-binding fragment to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody or antigen-binding fragment to the epi tope.
Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. A binding molecule can. be said to competitively inhibit .10 binding of the reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
[00871 As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual epitope with one or more antigen-binding domains, e.g., of an immunoglobulin molecule. See, e.g., Harlow et at., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of antigen-binding domains and an antigen. See, e.g., Harlow at pages 29-34.
Avidity is related to both the affinity of individual antigen-binding domains in the population with specific cpitopcs, and also the valencies of the inununoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity. An interaction between a bivalent monoclonal antibody with a receptor present at a high density on a cell surface would also be of high avidity.
[00881 Binding molecules, e.g., antibodies or fragments, variants, or derivatives thereof as disclosed herein can also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances.
Thus, a binding molecule is cross reactive if it binds to an epitope other than the one that induced its formation. The cross-reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original.

- 26 -A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof can also be described or specified in tem-is of their binding affinity to an antigen. For example, a binding molecule can bind to an antigen with a dissociation constant or KD
no greater than 5 x 10-2 M, 10-2 M, 5 x 10-3 M, 10-3 M, 5 x 10-4 M, 10-4 M, 5 x 10-5 M, 10-5 M, 5 x 10-6 M, 1.0-6 M, 5 x 10-7 M, 10-7 M. 5 x 10-8 M, 10-8 M, 5 x 10-9 M, 10-9 M, 5 x 10-10 M. 10-10 M, 5 x 10-11 M, 10-11 M, 5 x 10-12 M, 10-12 M, 5 x 10-13 M. 10-13 M, 5 x 10-14 M, 10-14 M, 5 x 10-15 M, or 10-15 M.

"Antigen-binding antibody fragments" including single-chain antibodies or other antigen-binding domains can exist alone or in combination with one or more of the .10 following: hinge region, CHI, CH2, CH3, or CH4 domains, J-chain, or secretory component. Also included are antigen-binding fragments that can inch ide any combination of variable region(s) with one or more of a hinge region, CHI, CH2, CH3, or domains, a 3-chain., or a secretory component. Binding molecules, e.g., antibodies, or antigen-binding fragments thereof can be from any animal origin including birds and mammals. The antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be condricthoid in origin (e.g., from sharks). As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgcnic for one or more human immunoglobulints and can in some instances express endogenous irnmunoglobulins and some not, as described infra and, for example in, U.S.
Pat. No.
5,939,598 by Kucherlapati et al. According to embodiments of the present disclosure, an IgM antibody, IgM-like antibody, or other IgM-derived binding molecule as provided herein can include an antigen-binding fragment of an antibody, e.g., a say fragment, so long as the IgM antibody, IgM-like antibody, or other IgM-derived binding molecule is able to form a multimen. e.g., a hemmer or a pentamer, and an IgA antibody, IgA-like antibody, or other IgA-derived binding molecule as provided herein can include an antigen-binding fragment of an antibody, e.g., a scFv fragment, so long as the IgA
antibody, IgA-like antibody, or other IgA-derived binding molecule is able to form a multimer, e.g., a dimer. As used herein such a fragment comprises a "multimeiizing fragment."

As used herein, the term "heavy chain subunit" includes amino acid sequences derived from an imrnunoglobul in heavy chain, a binding molecule, e.g., an antibody,

- 27 -antibody-like, or antibody-derived molecule comprising a heavy chain subunit can include at least one of. a VH domain, a CH I domain, a hinge (e.g., upper. middle, and/or lower hinge region) domain, a CH2 domain, a C143 domain, a CH4 domain, or a variant or fragment thereof. For example, a binding molecule, e.g., an antibody, antibody-like, or antibody-derived molecule, or fragment, e.g., multimerizing fragment, variant, or derivative thereof can include without limitation, in addition to a domain:, a CHI
domain; a CHI domain, a hinge, and a CH2 domain; a CHI domain and a CH3 domain; a CHI domain, a hinge, and a CH3 domain; or a CHI domain, a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments a binding molecule, e.g., an.
antibody, antibody-like, or antibody-derived molecule, or fragment, e.g., multimerizing fragment, variant, or derivative thereof can include, in addition to a VH domain, a CH3 domain and a CH4 domain; or a CH3 domain, a CH4 domain, and a J-chain. Further, a binding molecule, e.g., an antibody, antibody-like, or antibody-derived molecule, for use in the disclosure can lack certain constant region portions, e.g., all or part of a C112 domain. It will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain subunit) can be modified such that they vary in amino acid sequence from the original immunoglobulin molecule. According to embodiments of the present disclosure, an IgM antibody, IgM-like antibody, or other IgM-derived binding molecule as provided herein comprises sufficient portions of an IgM heavy chain constant region to allow the IgM antibody, IgM-like antibody, or other IgM-derived binding molecule to form a multimer, e.g., a hexamer or a pentamer. As used herein such a fragment comprises a "multimerizing fragment."

As used herein, the term "light chain subunit" includes amino acid sequences derived from an immunoglobulin light chain. The light chain subunit includes at least a VL, and can further include a CL (e.g., Ct: or CX) domain.

Binding molecules, e.g., antibodies, antibody-like molecules, antibody-derived molecules, antigen-binding fragments, variants, or derivatives thereof, or multimerizing fragments thereof can be described or specified in terms of the epitope(s) or portion(s) of a target, e.g., a target antigen that they recognize or specifically bind. The portion of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an "epitope," or an "antigenic determinant." A target antigen can comprise a single epitope or at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.

- 28 -100941 As used herein the term "disulfide bond" includes the covalent bond formed between two sulfur atoms, e.g., in cysteine residues of a poly-peptide. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
Disulfide bonds can be "intra-chain," i.e., linking to cysteine residues in a single polypeptide or polypeptide subunit, or can be "inter-chain," i.e., linking two separate polypeptide subunits, e.g., an antibody heavy chain and an antibody light chain, to antibody heavy chains, or an IgM or IgA antibody heavy chain constant region and a J-chain.

As used herein, the term. "chimeric antibody" refers to an antibody in which the .10 immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial or modified) is obtained from a second species. In some embodiments the target binding region or site will be from a non-human source (e.g. mouse or primate) and the constant region is human.

The terms "multispecific antibody" or "bispecific antibody" refer to an antibody, antibody-like, or antibody-derived molecule that has antigen-binding domains for two or more different epitopes within a single antibody molecule. Other binding molecules in addition to the canonical antibody structure can be constructed with two binding specificities. Epitope binding by bispecific or multispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas arc two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Strohlein and Fleiss, Future Oncol. 6:1387-94(2010); Mabry and Snavely, !Drugs.
13:543-9 (2010)). A bispecific antibody can also be a diabody.

As used herein, the term "engineered antibody" refers to an antibody in which a variable domain, constant region, and/or J-chain is altered by at least partial replacement of one or more amino acids. In certain embodiments entire CDRs from an antibody of known specificity can be grafted into the framework regions of a heterologous antibody.
Although alternate CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, CDRs can also be derived from an antibody of different class, e.g., from an antibody from a different species.
An engineered antibody in which one or more "donor" CDRs from. a non-human antibody of known specificity are grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody." In certain embodiments not all of the CDRs are replaced with the complete CDRs from the donor variable region and yet the antigen-

- 29 -binding capacity of the donor can still be transferred to the recipient variable domains.
Given the explanations set forth in, e.g., U. S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing, to obtain a functional engineered or humanized antibody.

As used herein the term "engineered" includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, nucleic acids, or glycans, or some combination of these techniques).
.10 100991 As used herein, the terms "linked," "fused" or "fusion" or other grammatical equivalents can be used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more poly-nucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing, two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, in-frame linker sequence. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.

In the context of polypeptides, a "linear sequence" or a "sequence" is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which amino acids that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A portion of a polypeptide that is "amino-terminal" or "N-terminal" to another portion of a polypeptide is that portion that comes earlier in the sequential polypeptide chain. Similarly, a portion of a polypeptide that is "earboxy-terminal" or "C-terminal" to another portion of a polypeptide is that portion that comes later in the sequential poly-peptide chain. For example, in a typical antibody, the variable domain is "N-terminal" to the constant region, and the constant region is "C-terminal"
to the variable domain.

- 30 -101011 The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into RNA, e.g., messenger RNA (mRNA), and the translation of such niRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors.
Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide that is .10 translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate"
refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt or slow the progression of an existing diagnosed pathologic condition or disorder. Terms such as "prevent," "prevention," "avoid," "deterrence" and the like refer to prophylactic or preventative measures that prevent the development of an undiagnosed targeted pathologic condition or disorder. Thus, "those in need of treatment" can include those already with the disorder and/or those prone to have the disorder.

As used herein the terms "serum half-life" or "plasma half-life" refer to the time it takes (e.g., in minutes, hours, or days) following administration for the serum or plasma concentration of a drug, e.g., a binding molecule such as an antibody, antibody-like, or antibody-derived molecule or fragment, e.g., multimerizing fragment thereof as described herein, to be reduced by 50%. Two half-lives can be described: the alpha half-life; a half-life, or t1/2a, which is the rate of decline in plasma concentrations due to the process of drug redistribution from the central compartment, e.g., the blood in the case of intravenous delivery, to a peripheral compartment (e.g., a tissue or organ), and the beta half-life, 11 half-life, or t1/213 which is the rate of decline due to the processes of excretion or metabolism.
101041 As used herein the term "area under the plasma drug concentration-time curve" or "AUC" reflects the actual body exposure to drug after administration of a dose of the drug and is expressed in mg*h/L. This area under the curve can be measured, e.g., from time 0

- 31 -(t0) to infinity (co) and is dependent on the rate of elimination of the drug from the body and the dose administered.
[01051 As used herein, the term "mean residence time" or "MRT" refers to the average length of time the drug remains in the body.
101061 By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject. in certain embodiments the subject is a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on..
101071 As used herein, as the term "a subject that would benefit from therapy" refers to a subset. of subjects, from amongst all prospective subjects, which would benefit from administration of a given therapeutic agent, e.g., a binding molecule such as an antibody, comprising one or more antigen-binding domains. Such binding molecules, e.g., antibodies, can be used, e.g., for a diagnostic procedure and/or for treatment or prevention of a disease.
PD-1 binding molecules [01081 Provided herein are multimeric binding molecules comprising two, five, or six bivalent binding units or variants or fragments thereof, wherein each binding unit comprises two IgA or IgM heavy chain constant regions or multimerizing fragments or variants thereof; each associated with a binding domain, wherein three to twelve of thc binding domains are programmed cell death protein I (PD-1)-binding domains that specifically and agonistically bind to PD-1. In certain embodiments, the binding molecule can activate PD-1-mediated signal transduction in a cell at a higher potency than an equivalent amount of a bivalent IgG antibody or fragment thereof comprising two of the same PD-1-binding domains, which also specifically binds to and agonizes PD-1.
In some embodiments, the two, five, or six binding units are human, humanized, or chimeric immunoglobulin binding units. The provided binding molecules can be used as therapeutics or diagnostics, e.g., to treat mitoimmune disorders.

In some embodiments, the multimeric binding molecules are dimeric and comprise two bivalent binding units or variants or fragments thereof. In some embodiments, the multimeric binding molecules are dimeric, comprise two bivalent binding units or variants or fragments thereof, and further comprise a i-chain or functional fragment or variant

-32 -thereof as described herein. In some embodiments, the multimeric binding molecules are dimeric, comprise two bivalent binding units or variants or fragments thereof, and fiirther comprise a J-chain or functional fragment or variant thereof as described herein, wherein each binding unit comprises two IgA heavy chain constant regions or multimerizing fragments or variants thereof.

In some embodiments, the multimeric binding molecules are pentameric and comprise five bivalent binding units or variants or fragments thereof. In some embodimentsõ the multimeric binding molecules are pentameric and comprise five bivalent binding units or variants or fragments thereof, and further comprise a J-chain or functional fragment or variant thereof as described herein. In some embodiments, the multimeric binding molecules are pentametic and comprise five bivalent binding units or variants or fragments thereof, and further comprise a J-chain or functional fragment or variant thereof as described herein, wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof.

In some embodiments, the multimeric binding molecules are hexameric and comprise six bivalent binding units or variants or fragments thereof. In some embodiments, the multimeric binding molecules are hexarneric and comprise six bivalent binding units or variants or fragments thereof, and wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments or variants thereof In certain embodiments, heavy chain constant regions in the provided binding molecules are each associated with a binding domain, e.g., an antibody antigen-binding domain, e.g., a scFv, a VHH or the VH subunit of an antibody antigen-binding domain.
The multimeric binding molecule discloses herein can comprise three to twelve binding domains that are programmed cell death protein I (PD- .1)- binding domains that specifically and agonistically bind to PD-1. In some embodiments, the multimeric binding molecule, such as an IgA antibody, an IgA-like antibody, or an IgA-derived binding molecule comprises three or four binding domains that specifically and agonistically bind to PD-1. In some embodiments, the multimeric binding molecule, such as an IgA
antibody, an IgA-like antibody, or an IgA-derived binding molecule comprises four binding domains that specifically and agonistically bind to PD-1. In some embodiments, the multimeric binding molecule, such as an IgM antibody, an IgM-like antibody, or an IgM-derived binding molecule comprises ten or twelve binding domains that specifically and agonistically bind to PD-1.

- 33 -In certain embodiments, the provided multimeric binding molecule is multispecific, e.g., bispecific, trispecific, or tetraspecifie, where two or more binding domains associated with the heavy chain constant regions of the binding molecule specifically bind to different targets. In certain embodiments, the binding domains of the multimeric binding molecule all specifically bind to PD-1. In certain embodiments, the binding domains of the multimeric binding molecule are identical. in such cases, the multimeric binding molecule can still be bispecific, if, for example, a binding domain with a different specificity is part of a modified J-chain as described elsewhere herein. In certain embodiments, the binding domains are antibody-derived antigen-binding domains, e.g., a scFv associated with the .10 heavy chain constant regions or a VII subunit of an antibody binding domain associated with the heavy chain constant regions.

In certain embodiments, each binding unit comprises two heavy chains each comprising a VH situated amino terminal to the heavy chain constant region, and two immunoglobulin light chains each comprising alight chain variable domain (VL) situated amino terminal to an iinmunoglobulin light chain constant region, e.g., a kappa or lambda constant region. The provided VII and VL combine to form an antigen-binding domain that specifically binds to the target. In certain embodiments each antigen-binding domain of each binding molecule binds to the same target, i.e., PD-1. In certain embodiments, each antigen-binding domain of each binding molecule is identical.
101151 In certain embodiments, the three to twelve PD- 1-binding domains of the multimeric binding molecule comprise a heavy chain variable region (VII) and a light chain variable region (VL), wherein the VH and VL comprise six immtmoglobulin complementarity determining regions HCDR1, HCDR2, HCDR.3, LCDRI, LCDR2, and LCDR3, wherein the EICDR1, HCDR2, HCDR3, LCDR.I. LCDR2, and LCDR3 comprise the CDRs of an antibody comprising the VH and VL of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO:
3 and SEQ ID NO: 4, SEQ Ill NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID
NO:
8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO:

13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ
ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or the VH of any one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID
NO:
21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VL of any one of SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22, or the CDRs of an antibody comprising

- 34 -the VH and VL of SEQ ID NO: I and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO:
14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID
NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively with one or two single amino acid substitutions in one or more of the HCDIts or LCDIts, or the VH of any one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO:
23, or SEQ ID NO: 24 and the VI. of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ
ID NO: 20, or SEQ ID NO: 22 with one or two single amino acid substitutions in one or more of the HCDR s or I ,CDRs, such as the CDRs of an antibody compiising the VI-I and VL of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO:

13 and SEQ ID NO: 14, and SEQ ID NO: 25 and SEQ ID NO: 26, respectively, or the CDRs of an antibody comprising the VII and VL of SEQ ID NO: 1 and SEQ ID NO:
2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 13 and SEQ ID NO: 14, and SEQ ID NO:
and SEQ ID NO: 26, respectively with. one or two single amino acid substitutions in one or more of the HCDRs or LCDRs.
(01161 In certain embodiments, the three to twelve PD-1-binding domains of the multimeric binding molecule comprise an antibody VH and a VL, wherein the VH and VL
comprise 20 amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100%
identical to SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ

ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ 11) NO: 9 and SEQ
ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO:
25 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ
ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or the VH of any one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ

ID NO: 24 arid the VI. of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:
20, or SEQ ID NO: 22, such as the amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID
NO: 3 and SEQ T.D NO: 4, SEQ ID NO: 13 and SEQ ID NO: 14, and SEQ ID NO: 25 and SEQ
ID NO: 26, respectively.

- 35 -In certain embodiments, the three to twelve PD-1-binding domains comprise antibody VH and VI, regions comprising the amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ TD NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID
NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ
ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or the VH of any one of SEQ ID NO: 15, SEQ ID
NO:
17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24 and the VI, of any .10 one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22, such as VH
and VI, regions comprising the amino acid sequences SEQ ID NO: 1 and SEQ ID
NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO:
25 and SEQ ID NO: 26, respectively.
101.181 In certain embodiments, each binding unit of the multimeric binding molecule comprises two heavy chains and two light chains, wherein the heavy chains and light chains comprise VH and VIõ amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ Ill NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO:
13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ
ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or the VH of any one of SEQ ID NO: 15, SEQ TD NO: 17, SEQ ID NO: 19, SEQ ID
NO:
21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VL of any one of SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22, such as VH and VL amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 13 and SEQ ID NO:
14, or SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

In certain embodiments, the heavy chains and light chains of the multimeric binding molecule comprise the VH and VL amino acid sequences SEQ ID NO: I and SEQ ID
NO:
2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO:

12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID

- 36 -NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID
NO: 50, respectively, or the VU of any one of SEQ TD NO: 15, SEQ ID NO: 17, SEQ ID
NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VL of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22, such as the VH
and VL amino acid sequences SEQ ID NO: I and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ
ID NO: 4, SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO: 25 and SEQ ID NO: 26, respectively.
IgM antibodies, IgM-like antibodies, other IgM-derived binding molecules 101201 IgM is the first inununoglobulin produced by B cells in response to stimulation by antigen. Naturally-occurring IgM is naturally present at around 1.5 mg/ml in serum with a half-life of about 5 days. IgM is a pentameric or hexameric molecule and thus includes five or six binding units. An IgM binding unit typically includes two light and two heavy chains. While an IgG heavy chain constant region contains three heavy chain constant domains (CHI, CH2 and CH3), the heavy (p) constant region of IgM additionally contains a fourth constant domain (CH4) and includes a C-terminal "tailpiece." The human IgM
constant region typically comprises the amino acid sequence SEQ ID NO: 35 (identical to, e.g., GenBank Accession Nos. pirilS37768, CAA47708.1, and . CAA47714.I, allele IGHM*03) or SEQ ID NO: 36 (identical to, e.g., GenBank Accession No.
spIP01871.4, allele 1GHM*04). The human Cpl region ranges from about amino acid 5 to about amino acid 102 of SEQ ID NO: 35 or SEQ ID NO: 36; the human Cp2 region ranges from about amino acid 114 to about amino acid 205 of SEQ ID NO: 35 or SEQ ID NO: 36, the human Cp3 region ranges from about amino acid 224 to about amino acid 319 of SEQ ID
NO: 35 or SEQ ID NO: 36, the Cp.i 4 region ranges from about amino acid 329 to about amino acid 430 of SEQ ID NO: 35 or SEQ ID NO: 36, and the tailpiece ranges from about amino acid 431 to about amino acid 453 of SEQ ID NO: 35 or SEQ ID NO: 36.
101211 Other forms and alleles of the human IgM constant region with minor sequence variations exist, including, without limitation, GenBank Accession Nos.
CAB37838. I , and pirlIMHHU. The amino acid substitutions, insertions, and/or deletions at positions con-esponding to SEQ ID NO: 35 or SEQ ID NO: 36 described and claimed elsewhere in this disclosure can likewise be incorporated into alternate human IgM
sequences, as well as into IgM constant region amino acid sequences of other species.

- 37 -Each IgM heavy chain constant region can be associated with a binding domain, e.g., an antigen-binding domain, e.g., a scFy or VHH, or a subunit of an antigen-binding domain, e.g., a VFI region. Exemplary antigen-binding domains, e.g., binding domains that specifically and agonistically bind PD-I are described elsewhere herein. In certain embodiments the binding domain can be a non-antibody binding domain, e.g., a receptor ectodomain, a ligand or receptor-binding fragment thereof, a cytokine or receptor-binding fragment thereof, a growth factor or receptor binding fragment thereof, a neurotransmitter or receptor binding fragment thereof, a peptide or protein hormone or receptor binding fragment thereof, an immune checkpoint modulator ligand or receptor-binding fragment .10 thereof, or a receptor-binding fragment of an extracellular matrix protein.
See, e.g., PCT
Application No. PCT U52019/057702, which is incorporated herein by reference in its entirety.

Five IgM binding units can form a complex with an additional small polypeptide chain (the J-chain), or a functional fragment, variant, or derivative thereof, to form a pentameric IgM antibody or IgM-like antibody, as discussed elsewhere herein.
The precursor form of the human J-chain is presented as SEQ ID NO: 40. The signal peptide extends from amino acid 1 to about amino acid 22 of SEQ ID NO: 40, and the mature human J-chain extends from about amino acid 23 to amino acid 159 of SEQ ID NO:
40.
The mature human I-chain includes the amino acid sequence SEQ ID NO: 41.
101241 Exemplary variant and modified 1-chains are provided elsewhere herein. Without the J-chain, an IgM antibody or IgM-like antibody typically assembles into a hexamer, comprising up to twelve antigen-binding domains. With a I-chain, an IgM
antibody or IgM-like antibody typically assembles into a pentamer, comprising up to ten antigen-binding domains, or more, if th.e J-chain is a modified .J-chain comprising one or more heterologous polypeptides comprising additional antigen-binding domain(s). The assembly of five or six 104 binding units into a pentameric or hexameric IgM
antibody or IgM-like antibody is thought to involve the Cp4 and tailpiece domains. See, e.g., Braathen, R., et al. , J Biol. Chem. 277:42755-42762 (2002). Accordingly, a pentameric or hexameric IgM antibody provided in this disclosure typically includes at least the Cp4 and tailpiece domains (also referred to herein collectively as Cp4-tp). A "multimerizing fragment" of an IgM heavy chain constant region thus includes at least the Cp4-tp domains.
An IgM
heavy chain constant region can additionally include a C1i3 domain or a fragment thereof, a C1.t2 domain or a fragment thereof, a CR1 domain or a fragment thereof, and/or other

- 38 -IgM heavy chain domains. In certain embodiments, an IgM-derived binding molecule, e.g., an IgM antibody, IgM-like antibody, or other igM-derived binding molecule as provided herein can include a complete IgM heavy (g) chain constant domain, e.g., SEQ
ID NO: 35 or SEQ ID NO: 36, or a variant derivative, or analog thereof, e.g., as provided herein.

In certain embodiments, the disclosure provides a multimeric binding molecule, e.g., pentameric or hexameric binding molecule, where the binding molecule includes ten or twelve IgM-derived heavy chains, and where the IgM-derived heavy chains comprise IgM
heavy chain constant regions each associated with a binding domain that specifically binds .10 to a target. In certain embodiments, the disclosure provides an IgM antibody, IgM-like antibody, or IgM-derived binding molecule that includes five or six bivalent binding units, where each binding unit includes two IgM or IgM-like heavy chain constant regions or multimerizing fragments or variants thereof, each associated with an antigen-binding domain or subunit thereof. In certain embodiments, the two IgM heavy chain constant regions included in each binding unit are human heavy chain constant regions.
In some embodiments, the heavy chains are elycosylated. In some embodiments, the heavy chains can be mutated to affect glycosylation.

Where the IgM antibody, IgM-like antibody, or other IgM-derived binding molecule provided in this disclosure is pcntarncric, the IgM antibody, IgM-like antibody, or other IgM-derived binding molecule typically further include a J-chain, or functional fragment or variant thereof. In certain embodiments, the J-chain is a modified J-chain or variant thereof that further comprises one or more heterologous moieties attached to the I-chain, as described elsewhere herein. In certain embodiments, the J-chain can be mutated to affect, e.g., enhance, the serum half-life of the IgM antibody, IgM-like antibody, or other IgM-derived binding molecule provided herein, as discussed elsewhere in this disclosure.
In certain embodiments the J-chain can be mutated to affect glycosylation, as discussed elsewhere in this disclosure.

An IgM heavy chain constant region can include one or more of a C pi I
domain or fragment or variant thereof, a Cp2 domain or fragment or variant thereof, a CP
domain or fragment or variant thereof, and/or a Cp4 domain or fragment or variant thereof, provided that the constant region can serve a desired function in the IgM
antibody, IgM-like antibody, or other IgM-derived binding molecule, e.g., associate with second IgM
constant region to form a binding unit with one, two, or more antigen-binding domain(s),

- 39 -and/or associate with other binding units (and in the case of a pentamer, a J-chain) to form a hexamer or a pentamer. in certain embodiments the two IQM heavy chain constant regions or fragments or variants thereof within an individual binding unit each comprise a C1u4 domain or fragment or variant thereof, a tailpiece (tp) or fragment or variant thereof, or a combination of a C1t4 domain and a TP or fragment or variant thereof. In certain embodiments the two WA- heavy chain constant regions or fragments or variants thereof within an individual binding unit each further comprise a C1.t3 domain or fragment or variant thereof, a C112 domain or fragment or variant thereof, a Cttl domain or fragment or variant thereof, or any combination thereof.
.10 [01281 In some embodiments, the binding units of the IgM antibody, IgM-like antibody, or other IgM-derived binding molecule comprise two light chains. In some embodiments, the binding units of the IgM antibody, IgM-like antibody, or other IgM-derived binding molecule comprise two fragments light chains. In some embodiments, the light chains are kappa light chains. In some embodiments, the light chains are lambda light chains. In some embodiments, each binding unit comprises two immunoglobulin light chains each comprising a VI., situated amino terminal to an immunoglobulin light chain constant region.
IgA antibodies, IgA-like antibodies, other IgA-derived binding molecules [01291 IgA. plays a critical role in muwsal immunity and comprises about 15% of total.
immunoglobulin produced. IgA is a monomeric or dimcric molecule. An IgA
binding unit includes two light and two heavy chains. IgA contains three heavy chain constant domains (Cal, Ccr2 and Ca3), and includes a C-terminal "tailpiece "Human IgA. has two subtypes, IgA 1 and IgA2. The human IgAl constant region typically includes the amino acid sequence SEQ ID NO: 37. The human Cal domain extends from about amino acid 6 to about amino acid 98 of SEQ ID NO: 37; the human IgAl hinge region extends from about amino acid 102 to about amino acid 124 of SEQ ID NO: 37, the human Ca3 domain extends from about amino acid 228 to about amino acid 330 of SEQ ID NO: 37, and the tailpiece extends from about amino acid 331 to about amino acid 352 of SEQ ID
NO: 37.
The human IgA2 constant region can include the amino acid sequence SEQ ID NO:
38, SEQ ID NO: 48, or other related isoforms known to those of skill in the art.
The human Cal domain extends from about amino acid 6 to about amino acid 98 of SEQ ID
NO: 38 or SEQ ID NO: 48; the human IgA2 hinge region extends from about amino acid 102 to

- 40 -about amino acid 111 of SEQ ID NO: 38 or SEQ ID NO: 48, the human Ca2 domain extends from about amino acid 113 to about amino acid 206 of SEQ ID NO: 38 or SEQ
ID NO: 48, the human Ca3 domain extends from about amino acid 215 to about amino acid 317 of SEQ ID NO: 38 or SEQ ID NO: 48, and the tailpiece extends from about amino acid 318 to about amino acid 340 of SEQ ID NO: 38 or SEQ ID NO: 48. SEQ ID
NOS:
37 and 38 are presented below:
SEQ ID NO: 37 (human IgAl constant region) ASPTSPKVFPLSLCSTQPDGN VVIACLV QGFEPQEPLSVT
WSESGQGVTARNEPPSQDASGDLYTTSSQLTLPATQCLA
GKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPS
PSCCIIPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGV
TFTWTPSSGK SAVQGPPERDLCGCYSVSSVLPGCAEPW
NI-IG KTFTCTAAYPESKTPLTATLSK SGNTFRPEVI-ILLPPP
SEELALNELVILTCLARGESPKDVINRWLQGSQELPREK
YLTWASRQEPSQUITI'EAVTSILRVAAEDWKKGDIFSC
MVGHEALPLAI4TQKTIDRLAGKETHVNVSVV1VIAE'VDG-TCY
SEQ ID NO: 38 (human IgA2 constant region) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSV
TWSESGQNVTARNEWSQDASGDLYTTSSQLTLPATQCP
DGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCITPRISLH
RPALEDLLLGSEANLTCTLTGLRDASGA.TFTWTPSSGK.S
AVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAA
HPELKTPLTANITKSGNTERPEVHLLPPPSEELALNELVT
LTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEP
SQGTITFAVTSILRVAAEDWKKGDTFSCMVGHEALPLA
FTQKTIDRMAGKP'TFIVNVSVVMAEVDGTCY
101301 Two IgA binding units can form a complex with two additional polypeptide chains, the J-chain (e.g., SEQ ID NO: 41 or SEQ ID NO: 42 and the secretory component (precursor, SEQ ID NO: 39, mature: amino acids 19 to 6(13 of SEQ ID NO: 39) to fonn a secretory IgA (sIgA) antibody. The assembly of IgA binding units into a dimeric sIgA
antibody is thought to involve the Ca3 and tailpiece domains (also referred to herein collectively as the Ca3-tp domain). Accordingly, a dimeric sIgA antibody provided in this disclosure typically includes IgA constant regions that include at least the Ca3 and tailpiece domains. SEQ ID NO: 39 is presented below:
SEQ ID NO: 39:
MLLINLTCLL A VFPAISTKSPIFGPEEVNSVEGNSVSITCY
YPPTSVNRHTRKYWCRQGARGGCITIJSSEGYVSSKYAG

- 41 -RANLTNFPENGTFV'VNIAQLSQDDSGRYKCGLGINSRGL
SFDVSLEVSQGPGLLNDTKVYTVDLGRTVTINCPFKIEN
AQICR_KSLYKQIGLYPVLVIDSSGYVNPNYTGRIRLDIQG
TGQLLESVVINQLRLSDAGQYLCQAGDDSNSNKKNADL
QVLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCRQ
SSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVI
TGLRKEDAGRYLCGAHSDGQLQEGSPIQAWQLFVNEES

GAQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTFTV
ILNQLTSRDAGFYW'CLTNGDTLWRITVEIKIIEGEPNLK
VPGNVTAVLCiETLKVPCITFPCKFSSYEKYWCKWNNTG
CQALPSQDEGPSKAFVNCDENSRLVSLTLNLVTRADEG
WYWCGVKQGHFYGETAA.VYVAVEERKAAGSRDVSLA.
KADAAPDEKVLDSGFREIENKAIQDPRLFAEEKAVADIR

GAVAVGVAR ARHRKNVDRVSIRSYRTDISMSDFF.NSRE
FGANDNMGASSITQETSLGGKEEFVATTESTTETKEPKK
AKR.SSKEEAEMAYKDFLLQSSTVAAEAQDGPQEA
101311 An IgA heavy chain constant region can additionally include a Ca2 domain or a fragment thereof, an IgA hinge region. a Cal domain or a fragment thereof, and/or other IgA heavy chain domains. In certain aspects, an IgA antibody or IgA-like binding molecule as provided herein can include a complete IgA heavy (a) chain constant domain (e.g., SEQ ID NO: 37 or SEQ ID NO: 38), or a variant, derivative, or analog thereof. In some embodiments, the IgA heavy chain constant regions or multimcrizing fragments thereof are human IgA constant regions.
101321 In some embodiments, the binding units of the IgA antibody, IgA-like antibody, or other IgA-derived binding molecule comprise two light chains. In some embodiments, the binding units of the IgA antibody, IgA-like antibody, or other IgA-derived binding molecule comprise two fragments light chains. In some embodiments, the light chains are kappa light chains. In some embodiments, the light chains are lambda light chains.
In some embodiments, each binding unit comprises two immunoglobulin light chains each comprising a VL situated amino terminal to an immunoglobulin light chain constant region.
J.-chains and functional fragments or variants thereof 101331 In certain embodiments, the multimerie binding molecule provided herein comprises a J -chain or functional fragment or variant thereof. In certain embodiments, the multimeric binding molecule provided herein, is pentameric and comprises a J-chain or functional

- 42 -fragment or variant thereof. In certain embodiments, the multimeric binding molecule provided herein is dimeric and comprises a J-chain or functional fragment or variant thereof. In some embodiments, the multimeric binding molecule can comprise a naturally occurring J-chain sequence, such as a mature human J-chain sequence (e.g., SEQ
ID NO:
41). Alternatively, in some embodiments, the multimeric binding molecule can comprise a variant J-chain sequence, such as a variant sequence described herein with reduced glycosylation or reduced binding to polymeric Ig receptor (e.g., plgR). In some embodiments., the multimeric binding molecule can comprise a functional fragment of a naturally occurring or variant J-chain. As persons of ordinary skill in the art will recognize, .10 "a functional fragment" or a "functional variant" in this context includes those fragments and variants that can associate with binding units; e.g., IgM or IgA heavy chain constant regions, to form a pentameric IgM antibody, IgM-like antibody, or IgM-derived binding molecule or a dimeric IgA antibody, IgA-like antibody, or IgA-derived binding molecule, and/or can associate with certain inun unoglobulin receptors, e.g., pIgR.
101341 In certain embodiments, the J-chain can be modified, e.g., by introduction of a heterologous moiety, or two or more heterologous moieties, e.g., polypeptides, without interfering with the ability of binding molecule to assemble and bind to its binding target(s). See U.S. Patent Nos. 9,951,134 and 10,400,038, and U.S. Patent Application Publication Nos. US-2019-0185570 and US-2018-0265596, each of which is incorporated herein by reference in its entirety.

Accordingly, a binding molecule provided by this disclosure, including multispecific IgA, IgA-like, IgM, or IgM-like antibodies as described elsewhere herein, can comprise a modified J-chain or functional fragment or variant thereof comprising a heterologous moiety, e.g., a heterologous polypeptide, introduced, e.g., fused or chemically conjugated, into the J-chain or fragment or variant thereof. In certain embodiments, the heterologous polypeptide can be fused to the N-terminus of the J-chain or functional fragment or variant thereof, the C-terminus of the J-chain. or functional fragment or variant thereof, or to both the N4erminus and C-term inns of the 3-chain or functional fragment or variant thereof In certain embodiments the heterologous polypeptide can be fused internally within the J-chain or functional fragment or variant thereof. In some embodiments, the heterologous poly-peptide can be introduced into the J-chain at or near a glycosylation site. In some embodiments, the heterologous polypeptide can be introduced into the J-chain within about 10 amino acid residues from the C-terminus, or within about 10 amino acids from

- 43 -the N-terminus. In certain embodiments, the heterologous polypeptide can be introduced into the mature human J-chain of SEQ ID NO: 41 between cysteine residues 92 and 101 of SEQ ID NO: 41, or an equivalent location in a J-chain sequence, e.g., a J-chain variant or functional fragment of a J-chain. In a further embodiment, the heterologous polypeptide can be introduced into the mature human J-chain of SEQ ID NO: 41 at or near a glycosylation site. In a further embodiment, the heterologous polypeptide can be introduced into the mature human J-chain of SEQ ID NO: 41 within about 10 amino acid residues from the C-terminus, or within about 10 amino acids from the N-terminus.

In certain embodiments the heterologous moiety can be a peptide or polypeptide .10 sequence fused in frame to the J-chain or chemically conjugated to the J-chain or fragment or variant thereof. In certain embodiments; the heterologous polypeptide is fused to the J-chain or functional fragment thereof via a peptide linker. Any suitable linker can be used, for example the peptide linker can include at least 5 amino acids, at least ten amino acids, and least 20 amino acids, at least 30 amino acids or more, and so on. In certain embodiments, the peptide linker includes least 5 amino acids, but no more than 25 amino acids. In certain embodiments the peptide linker can consist of 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, or 25 amino acids. In certain embodiments, the peptide linker consists of GGGGS (SEQ ID NO: 43), GGGGSGGGGS (SEQ ID NO: 44), GGGGSGGGGSGGGGS (SEQ ID NO: 45), GGGGSGGGGSGGGGSGGGGS (SEQ ID
NO: 46), or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 47).

In certain embodiments the heterologous moiety can be a chemical moiety conjugated to the J-chain. Heterologous moieties to be attached to a J-chain can include, without limitation, a binding moiety, e.g., an antibody or antigen-binding fragment thereof, e.g., a single chain Ey (scFv) molecule, a cytokine, e.g., IL-2 or IL-15 (see.
e.g., PCT
Application No. PCT US2019/057702, which is incorporated herein by reference in its entirety), a stabilizing peptide that can increase the half-life of the binding molecule, e.g., human serum albumin (HSA) or an HSA binding molecule, or a heterologous chemical moiety such as a polymer.

In some embodiments, a modified j-chain can comprise an antigen-binding domain that can include without limitation a polypeptide capable of specifically binding to a target antigen. In certain embodiments, an antigen-binding domain associated with a modified .1-chain can be an antibody or an antigen-binding fragment thereof. In certain embodiments the antigen-binding domain can be a scFv antigen-binding domain or a single-chain

- 44 -antigen-binding domain derived, e.g., from a camelid or condriethoid antibody.
In certain embodiments, the target is a target epitope, a target antigen. a target cell, or a target organ.
Targets can include, without limitation, auto-immune targets, immune checkpoint inhibitors, target antigens involved in blood-brain-barrier transport, target antigens involved in neurodegenerative diseases and neuroinflammatory diseases, and any combination thereof. In certain embodiments, the binding domain, e.g., scTv fragment can bind to an effector cell, e.g., a T cell or an NK cell. In certain embodiments, the binding domain, e.g., scFy fragment can specifically bind to CD3 on cytotoxic T cells, e.g., to CD3e. In some embodiments, the antigen binding domain binds ICOS Ligand (ICOSI,G), .10 e.g., UniProtKB-075144; ICOS (CD278), e.g., UniProtKB-Q9Y6W8; Interleukin 6(1L6), e.g., UniProtKR-P05231; CD28, e.g UniProtKR-P10747; CD3, e.g., C.D3E. or UniProtKB-P07766; CD80, e.g., UniProtK13-P33681; CD86, e.g., UniProtKB-P42081;

Tumor Necrosis Factor Alpha (TNFa), e.g., UniProtKB-P01375; or Fibroblast Activation Protein (FAP), e.g., UniProtKB-Q12884.
101391 The antigen-binding domain can be introduced into the 3-chain at any location that allows the binding of the antigen-binding domain to its binding target without interfering with J-chain function or the function of an associated multimeric binding molecule, e.g., a pentameric IgM or dimeric IgA antibody. Insertion locations include but are not limited to at or near the C-terminus, at or near the N-terminus or at an internal location that, based on the three-dimensional structure of the J-chain, is accessible.
Variant 3-chains that confer increased serum half-life In certain embodiments, the .1-chain is a functional variant 3-chain that includes one or more single amino acid substitutions, deletions, or insertions relative to a reference .1-chain identical to the variant .1-chain except for the one or more single amino acid substitutions, deletions, or insertions. For example, certain amino acid substitutions, deletions, or insertions can. result in the IgM-derived binding molecule exhibiting an increased serum half-life upon administration to a subject animal relative to a reference IgM-derived binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions in the variant 3-chain, and is administered using the same method to the same animal species. In certain embodiments the variant J-chain can include one, two, three, or four single amino acid substitutions, deletions, or insertions relative to the reference 3-chain.

- 45 -In certain embodiments, the j-chain, such as a modified j-chain, comprises an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J-chain (SEQ ID NO: 41). By "an amino acid corresponding to amino acid Y102 of the mature wild-type human J-chain" is meant the amino acid in the sequence of the J-chain, which is homologous to Y102 in the human J-chain. For example, see PCT Publication No. WO 2019/169314, which is incorporated herein by reference in its entirety. The position corresponding to Y102 in SEQ ID NO: 41 is conserved in the J-chain amino acid sequences of at least 43 other species. See FIG. 4 of U.S.
Patent No.
9,951,134, which is incorporated by reference herein. Certain mutations at the position .10 corresponding to Y102 of SEQ ID NO: 41 can inhibit the binding of certain immunoglobulin receptors, e.g., the human or marine Fcali receptor, the minim Fell receptor, and/or the human or murine polymeric 1g receptor (plgR) to an IgM
pentamer comprising the variant J-chain.

A multimeric binding molecule comprising a mutation at the amino acid corresponding to Y102 of SEQ ID NO: 41 has an improved serum half-life when administered to an animal than a corresponding multimeric binding molecule that is identical except for the substitution, and which is administered to the same species in the same manner. In certain embodiments, the amino acid corresponding to YI02 of SEQ ID
NO: 41 can be substituted with any amino acid. In certain embodiments, the amino acid corresponding to Y102 of SEQ ID NO: 41 can be substituted with alanine (A), serine (S) or arginine (R). In a particular embodiment, the amino acid corresponding to Y102 of SEQ
ID NO: 41 can be substituted with alanine. In a particular embodiment the J-chain or fiinctional fragment or variant thereof is a variant human J-chain referred to herein as and comprises the amino acid sequence SEQ ID NO: 42.
101431 Wild-type J-chains typically include one N-linked glycosylation site. In certain embodiments, a variant J-chain or functional fragment thereof of a multimeric binding molecule as provided herein includes a mutation within the asparagine(N)-linked glycosylation motif N-X 1 -SIT, e.g., starting at the amino acid position corresponding to amino acid 49 (motif N6) of the mature human J-chain (SEQ ID NO: 41) or J*
(SEQ 11.1 NO: 42), wherein N is asparagine, X1 is any amino acid except proline, and SIT
is serine or threon.ine, and wherein the mutation prevents glycosylation at that motif.
As demonstrated in PCT Publication No. WO 2019/169314, mutations preventing glycosylation at this site can result in the multimeric binding molecule as provided herein,

- 46 -exhibiting an increased serum half-life upon administration to a subject animal relative to a reference multimeric binding molecule that is identical except for the mutation or mutations preventing gly-cosylation in the variant J-chain, and is administered in the same way to the same animal species.
101441 For example, in. certain embodiments the variant J-chain or functional fragment thereof of a pentameric 1gM-derived or dirneric IgA-derived binding molecule as provided herein can include an amino acid substitution at the amino acid position corresponding to amino acid N49 or amino acid S51 of SEQ ID NO: 41 or SEQ ID NO: 42, provided that the amino acid corresponding to S51 is not substituted with threonine (T), or wherein the .10 variant J-chain comprises amino acid substitutions at the amino acid positions corresponding to both amino acids N49 and 551 of SEQ ID NO: 41 or SEQ ID NO:
42. In certain embodiments, the position corresponding to N49 of SEQ ID NO: 41 or SEQ
ID
NO: 42 is substituted with any amino acid, e.g., alanine (A), glycine (0), threonine (1), serine (S) or aspartic acid (D). In a particular embodiment, the position corresponding to N49 of SEQ ID NO: 41 or SEQ 1D NO: 42 can be substituted with alanine (A). In another particular embodimentõ the position corresponding to N49 of SEQ ID NO: 41 or SEQ ID
NO: 42 can be substituted with aspartic acid (D).
Variant 1gM constant regions 1gM heavy chain constant regions of a multinaeric binding molecule as provided herein can be engineered to confer certain desirable properties to the multimeric binding molecules provided herein. For example, in certain embodiments, 1gM heavy chain constant regions can be engineered to confer enhanced serum half-life to multimeric binding molecules as provided herein. Exemplary 1gM heavy chain constant region mutations that can enhance serum half-life of an 1gM-derived binding molecule are disclosed in PCT Publication No. WO 2019/169314, which is incorporated by reference herein in its entirety. For example, a variant 1gM heavy chain, constant region of the 1gM
antibody, IgM-like antibody, or 1gM-derived binding molecule as provided herein can include an amino acid substitution at a position corresponding to amino acid S401, E402, E403, R344, and/or E345 of a wild-type human 1gM constant region (e.g., SEQ ID
NO:
35 or SEQ ID NO: 36). By "an amino acid corresponding to amino acid S401, E402, E403, R344, and/or E345 of a wild-type human 1gM constant region" is meant the amino acid in the sequence of the 1gM constant region of any species which is homologous to S401,

- 47 -E402, E403, R344, and/or E345 in the human 1gM constant region. In certain embodiments, the amino acid corresponding to S401, F402, FA03, R344, and/or E345 of SEQ ID NO: 35 or SEQ ID NO: 36 can be substituted with any amino acid, e.g., alanine.
[01461 In certain embodiments, an 1gM antibody, 1gM-like antibody, or other 1gM-derived binding molecule as provided herein, can. be engineered to exhibit reduced complement-dependent cytotoxicity (CDC) activity to cells in the presence of complement, relative to a reference 1gM antibody, 1gM-like antibody, or other 1gM-derived binding molecule with corresponding reference human 1gM constant regions identical, except for the mutations conferring reduced CDC activity. These CDC mutations can. be combined with any of the .10 mutations to confer increased serum half-life as provided herein. By "corresponding reference human 1gM constant region" is meant a human 1gM constant region that is identical to the variant 1gM constant region except for the modification or modifications in the constant region affecting CDC activity. In certain embodiments, the variant human 1gM constant region includes one or more amino acid substitutions, e.g., in the Cp.3 domain, relative to a wild-type human 1gM constant region as described, e.g., in PCT
Publication No. WO/2018/187702, which is incorporated herein by reference in its entirety. Assays for measuring CDC are well known to those of ordinary skill in the art, and exemplary assays are described e.g., in PCT Publication No.
WO/2018/187702.
[01471 In certain embodiments, a variant htunan 1gM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human 1gM constant region at position L310, P311, P313, and/or K315 of SEQ ID NO: 35 (human 1gM constant region allele IGHM*03) or SEQ ID NO: 36 (human 1gM constant region allele IGHM*04). In certain embodiments, a variant human 1gM constant region conferring reduced CDC activity includes an amino acid substitution corresponding to the wild-type human 1gM constant region at position P311 of SEQ ID NO: 35 or SEQ
ID NO:
36. In other embodiments the variant 1gM constant region as provided herein contains an amino acid substitution corresponding to the wild-type human 1gM constant region at position P313 of SEQ ID NO: 35 or SEQ ID NO: 36. In other embodiments the variant 1gM constant region as provided herein contains a combination of substitutions corresponding to the wild-type human. 1gM constant region at positions P311 of SEQ ID
NO: 35 or SEQ ID NO: 36 and P313 of SEQ ID NO: 35 or SEQ ID NO: 36. These proline residues can be independently substituted with any amino acid, e.g., with alanine, serine, or glycine.

- 48 -101481 Human and certain non-human primate IgM constant regions typically include five (5) naturally-occurring asparagine (N)-linked glycosylation motifs or sites.
As used herein "an N-linked glycosylation motif" comprises or consists of the amino acid sequence N-X I-S/T, wherein N is asparagine, X1 is any amino acid except proline (P), and SIT is serine (S) or threonine (1). The elycan is attached to the nitrogen atom of the asparagine residue. See, e.g., Drickamer K, Taylor ME (2006), Introduction to Glycobiology (2nd ed.). Oxford University Press, USA. N-linked glycosylation motifs occur in the human IgM heavy chain constant regions of SEQ Ill NO: 35 or SEQ ID NO: 36 starting at positions 46 ("NI"), 209 ("N2"), 272 ("N3"), 279 ("N4"), and 440 ("N5"). These five .10 motifs are conserved in non-human primate IgM heavy chain constant regions, and four of the Five are conserved in the mouse IgM heavy chain constant region.
Accordingly, in some embodiments, IgM heavy chain constant regions of a multimeric binding molecule as provided herein comprise 5 N-linked glycosylation motifs: NI, N2, N3, N4, and N5. In some embodiments, at least three of the N-linked glycosylation motifs (e.g., Ni, N2, and N3) on each IgM heavy chain constant region are occupied by a complex glycan.

In certain embodiments, at least one, at least two, at least three, or at least four of the N- X I.-S/T motifs can include an amino acid insertion, deletion, or substitution that prevents glycosylation at that motif. In certain embodiments, the IgM-derived multimeric binding molecule can include an amino acid insertion, deletion, or substitution at motif NI, motif N2, motif N3, motif N5, or any combination of two or more, three or more, or all four of motifs Ni, N2, N3, or N5, where the amino acid insertion, deletion, or substitution prevents glycosylation at that motif. In some embodiment, the IgM
constant region comprises two or more substitutions relative to a wild-type human IgM
constant region at positions 46, 209, 272, or 440 of SEQ ID NO: 35 (human IgM constant region allele IGHM*03) or SEQ ID NO: 36 (human IgM constant region allele IGHM*04).
See, e.g., U.S. Provisional Application No. 62/891,263, which is incorporated herein by reference in its entirety.
Poly nucleotides and Vectors In certain embodiments, this disclosure provides a polynucleotide comprising a nucleic acid sequence that encodes a polypeptide subunit of a multimeric binding molecule described herein. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a heavy chain constant region and at least an antibody VII portion of the PD-

- 49 -1-binding domain of the multimeric binding molecule. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising the heavy chain of the multimeric binding molecule.
(01511 In some embodiments, the polynucleotide encodes a polypeptide subunit comprising a light chain constant region. and an antibody VI, portion of the PD-1-binding domain of the multimeric binding molecule. In some embodiments, the polynucleotide encodes a polypeptide subunit comprising the light chain of the multimeric binding molecule.

In certain embodiments, this disclosure provides a vector comprising one or more polynucleotides described herein. In some embodiments, the vector further comprises a .10 polynucleotide comprising a nucleic acid sequence that encodes a 3-chain or a functional fragment or variant thereof In certain embodiments, this disclosure provides a composition comprising a first vector and a second vector, wherein: a) the first vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the heavy chain of the multimeric binding molecule and the second vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the light chain, of the multimeric binding molecule, b) the first vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the heavy chain of the multimeric binding molecule and a polynucleotide comprising a nucleic acid sequence that encodes the light chain of the multimeric binding molecule and the second vector comprises a polynucleotide comprising a nucleic acid sequence that encodes a J-chain or a functional fragment or variant thereof, c) the first vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the heavy chain of the multimeric binding molecule and a polynucleotide comprising a nucleic acid sequence that encodes a J-chain or a functional fragment or variant thereof and the second vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the light chain of the multimeric binding molecule, or d) the first vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the light chain of the multimeric binding molecule and a polynucleotide comprising a nucleic acid sequence that encodes a T-chain or a functional fragment or variant thereof and the second vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the heavy chain, of the multimeric binding molecule. In certain embodiments, this disclosure provides a composition comprising a first vector, a second vector, and a third vector, wherein the first vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the

- 50 -heavy chain of the multimeric binding molecule, the second vector comprises a polynucleotide comprising a nucleic acid sequence that encodes the liQht chain of the mukimerie binding molecule, and the third vector comprises a polynucleotide comprising a nucleic acid sequence that encodes a I-chain or a functional fragment or variant thereof.
Host cells In certain embodiments, this disclosure provides a host cell that is capable of producing the multimeric binding molecule as provided herein, hi certain embodiments, the host cell comprises one or more vectors, a composition comprising multiple vectors, or polynucleotides disclosed herein. The disclosure also provides a method of producing the multimeric binding molecule as provided herein, where the method comprises culturing the provided host cell, and recovering the multimeric binding molecule.
Methods of Use 101551 The disclosure further provides a method of treating a disease or disorder in a subject in need of treatment, where the method includes administering to the subject a therapeutically effective amount of a multimeric binding molecule as provided herein. By "therapeutically effective dose or amount" or "effective amount" is intended an amount of a multimeric binding molecule that when administered brings about a positive immunotherapeutic response with respect to treatment of subject.

Effective doses of compositions for treatment of a disease or disorder vary depending upon many different factors, including means of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the subject is a human, but non-human mammals including transgenic mammals can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

In certain embodiments, the disclosure provides a method for treating an autoimmune disorder, an inflammatory disorder, or a combination thereof in a subject in need of treatment, where the method includes administering to the subject an effective amount of a multimeric binding molecule as provided herein. In certain embodiments, administration of a multimeric binding molecule as provided herein to a subject results in

-51 -greater potency than administration of an equivalent amount of a monomeric or dimeric binding molecule binding to the same binding partner. In certain embodiments the monomeric or dimeric binding molecule includes identical binding polypeptides to the multimeric binding molecule as provided herein. By "an equivalent amount" is meant, e.g., an amount measured by molecular weight, e.g., in total milligrams, or alternative, a molar equivalent, e.g., where equivalent numbers of molecules are administered.

In certain embodiments, the autoimmune disease can be, e.g., arthritis, e.g., rheumatoid arthritis, osteoarthritis, or ankylosing spondylitis, multiple sclerosis (MS), inflammatory bowel disease (1BD) e.g., Crohn's disease or ulcerative colitis, or systemic .10 lupus erythematosus (SLE). In certain embodiments the inflammatory disease or disorder n be, e.g., arthritis, e.g., rheumatoid arthritis, or osteoarthritis, or psoriatic arthritis,[.me disease, SLE, MS, Sjogren's syndrome, asthma, inflammatory bowel disease, ischemia, atherosclerosis, or stroke.

In other embodiments, the disclosure provides a method for preventing transplantation rejection in a transplantation recipient, where the method includes administering to the subject an effective amount of a multimeric binding molecule as provided herein. In certain embodiments, administration of a multimeric binding molecule as provided herein to a subject result in greater potency than administration of an equivalent amount of a monomeric or dimcric binding polypcptide binding to the same binding partner. In certain embodiments the monomeric or dimeric binding molecule includes identical binding polypeptides to the multimeric binding molecule as provided herein. By "an equivalent amount" is meant, e.g., an amount measured by molecular weight, e.g., in total milligrains, or alternative, a molar equivalent, e.g., where equivalent numbers of molecules are administered.
101601 The subject to be treated can be any animal, e.g., mammal, in need of treatment, in certain embodiments, the subject is a human subject.

In its simplest form, a preparation to be administered to a subject is mul.timeric binding molecule as provided herein administered in a conventional dosage form, which can be combined with a pharmaceutical excipient, carrier or diluent as described elsewhere herein.

A multimeric binding molecule ofthe disclosure can be administered by any suitable method, e.g., parenterally, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, bucmily, vaginally or via an implanted reservoir. The term "parenteral"

-52 -as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-sytiovial, intrasternal, intrathecal, intrahepatic, intralesional and intracmnial injection or infusion techniques.
Pharmaceutical Compositions and Administration Methods 101631 Methods of preparing and administering a multimeric binding molecule as provided herein to a subject in need thereof are well, known to or are readily determined by those skilled in the art in view of this disclosure. The route of administration of can be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While these forms of administration are contemplated as suitable forms, another example of a form for administration would be a solution for injection, in particular for intravenous, or intraarterial injection or drip.
A suitable pharmaceutical composition can include a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer agent (e.g., human albumin), etc.
(01641 As discussed herein, a multimeric binding molecule as provided herein can be administered in a pharmaceutically effective amount for the treatment of a subject in need thereof. In this regard, it will be appreciated that the disclosed multimeric binding molecule can be formulated so as to facilitate administration and promote stability of the active agent. Pharmaceutical compositions accordingly can include a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives, and the like. A pharmaceutically effective amount of a multimeric binding molecule as provided herein means an amount sufficient to achieve effective binding to a target and to achieve a therapeutic benefit. Suitable formulations are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed . (1980).

Certain pharmaceutical compositions provided herein can be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions, or solutions. Certain pharmaceutical. compositions also can. be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.

- 53 -101661 The amount of a multimeric binding molecule that can be combined with carrier materials to produce a single dosage form will vary depending, esz., upon the subject treated and the particular mode of administration. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).
[0167]
In keeping with the scope of the present disclosure, a multimetic binding molecule as provided herein can be administered to a subject in need of therapy in an amount sufficient to produce a therapeutic effect. A multimeric binding molecule as provided herein can be administered to the subject in a conventional dosage form prepared by combining the multimeric binding molecule of the disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known techniques.
The form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.

This disclosure also provides for the use of a multimeric binding molecule as provided herein in the manufacture of a medicament for treating, preventing, or managing a disease or disorder, e.g., an autoinunune disease, an inflammatory disease, or for preventing transplantation rejection.
101691 This disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Green and Sambrook, ed. (2012) Molecular Cloning A Laboratory Manual (4th ed.; Cold Spring Harbor Laboratory Press);
Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover and B.D. Hames, eds., (1995) DNA Cloning 2d Edition (IRL Press), Volumes 1-4; Gait, ed. (1990) Oligonucleotide Synthesis (IRL
Press); Mullis et al. IJ.S. Pat. No. 4,683,195; 1-lames and Higgins, eds. (1985) Nucleic Acid Hybridization (IRL Press); Haines and Higgins, eds. (1984) Transcription And Translation (IRL Press);
Freshney (2016) Culture Of Animal Cells, 7th Edition (Wiley-Blackwell);
Woodward, J., Immobilized Cells And Enzymes (IRL Press) (1985); Perbal (1988) A Practical Guide To Molecular Cloning; 2d Edition (Wiley-Interscience); Miller and Cabs eds.
(1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); S.C.
Malc rides

- 54 -(2003) Gene Transfer and Expression in Mammalian Cells (Elsevier Science);
Methods in Enzymology. Vols. 151-155 (Academic Press, Inc., N.Y.); Mayer and Walker, eds.
(1987) Immunochemical. Methods in Cell and Molecular Biology (Academic Press, London);
Weir and Blackwell, eds.; and in Ausubel et al. (1995) Current Protocols in Molecular Biology (John. Wiley and Sons).

General principles of antibody engineering are set forth, e.g., in Strohl, W.R., and L.M. Stroh' (2012), Therapeutic Antibody Engineering (Woodhead Publishing).
General principles of protein engineering are set forth, e.g., in Park and Cochran, eds. (2009), Protein Engineering and Design (CDC Press). General principles of iinmunology are set .10 forth, e.g., in: Abbas and Lichunan (2017) Cellular and Molecular Immunology 9th Edition (Elsevier) Additionally, standard methods in immunology known in the art can be followed, e.g., in Current Protocols in immunology (Wiley Online Library);
Wild, D.
(2013), The Immunoassay Handbook 4th Edition (Elsevier Science); Greenfield, ed.
(2013), Antibodies, a Laboratory Manual, 2d Edition (Cold Spring Harbor Press); and Ossipow and Fischer, eds., (2014), Monoclonal Antibodies: Methods and Protocols (Humana Press).

All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.
Exemplary Embodiments 101721 Among the provided embodiments arc:
101731 Embodiment I.
A multimeric binding molecule comprising two, five, or six bivalent binding units or variants or fragments thereof, 101741 wherein each binding unit comprises two IgA or IgM heavy chain constant regions or multimerizing fragments or variants thereof, each associated with a binding domain, 101751 wherein three to twelve of the binding domains arc programmed cell death protein 1 (PD-1)-binding domains that specifically and agonistically bind to PD-1, wherein the binding molecule can activate PD-1-mediated signal transduction in a cell at a higher potency than an equivalent amount of a bivalent IgG antibody or fragment thereof comprising two of the same PD-1-binding domains, which also specifically binds to and agonizes PD-1.

- 55 -

56 101771 Embodiment 2.
The multimeric binding molecule of embodiment 1, wherein the two, five, or six binding units are human, humanized, or chimeric imin unogl obul in binding units.
101781 Embodiment 3. The multimeric binding molecule of embodiment 1 or embodiment 2, wherein the three to twelve PD- I-binding domains comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH
and VL
comprise six immunoglobulin complementarity determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR I, HCDR2, HCDR3, LCDR I , LCDR2, and LCDR3 comprise the CDRs of an antibody comprising the VH and VI., of .10 SEQ TD
NO: land SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: Sand SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO:
25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ
ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or the VH of any one of SEQ ID
NO:
15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO:
24 and the VL of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ
ID NO: 22, or the CDRs of an antibody comprising the VH and VL of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ TD
NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ
ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively with one or two single amino acid substitutions in one or more of the HCDRs or LCDRs, or the VH of any one of SEQ ID NO: 15, SEQ ID
NO:
17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VL
of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22 with one or two single amino acid substitutions in one or more of the HCDRs or LCDRs.
101791 Embodiment 4. The multimeric binding molecule of embodiment 3, wherein the VII and VI. comprise six irn.munoglobulin complementarity determining regions HCDR I , HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1, T-ICDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the CDRs of an antibody comprising the VH and VI., of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID
NO:

13 and SEQ ID NO: 14, and SEQ ID NO: 25 and SEQ ID NO: 26, respectively, or the CDRs of an antibody comprising the VH and VI.: of SEQ ID NO: 1 and SEQ ID NO:
2, SEQ ID NO: 3 an.d SEQ ID NO: 4, SEQ ID NO: 13 and SEQ TD NO: 14, and SEQ ID
NO:
25 and SEQ ID NO: 26, respectively with one or two single amino acid substitutions in one or more of the HCDRs or LCDRs.
101801 Embodiment 5.
The multimeric binding molecule of any one of embodiments 1 to 3, wherein the three to twelve PD-1-binding domains of the binding molecule comprise an antibody VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO:
1 and SEQ
.10 ID NO:
2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: Sand SEQ ID NO: 6, SEQ ID
NO: 7 and SEQ in NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEX) ID NO: 11 and SEQ

ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO:
31 and SEQ TD NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ.
ID NO: 50, respectively, or the VH of any one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ
ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VI. of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22.
101811 Embodiment 6.
The multimeric binding molecule of embodiment 5, wherein the three to twelve PD-1-binding domains of the binding molecule comprise an antibody 'VH
and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%.. at least 90%, at least 95% or 100% identical to SEQ ID NO: 1 and SEQ ID
NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 13 and SEQ ID NO: 14, and SEQ ID NO:

and SEQ ID NO: 26, respectively.
101821 Embodiment 7.
The multimeric binding molecule of embodiment 5, wherein the 25 three to twelve PD-1-binding domains comprise antibody VH and VL regions comprising the amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ
ID
NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID
NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO:
28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID
NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or the VII of any one of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,

- 57 -SEQ ID NO: 23, or SEQ ID NO: 24 and the VL of any one of SEQ ID NO: 16, SEQ ID

NO: IS. SEQ ID NO: 20, or SEQ ID NO: 22.
(01831 Embodiment 8.
The multimeric binding molecule of embodiment 7, wherein the three to twelve PD-1-binding domains comprise antibody VH and VL regions comprising the amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ
ID
NO: 4, SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO: 25 and SEQ TD NO: 26, respectively.
101841 Embodiment 9. The multimeric binding molecule of any one of embodiments to 3, 5, or 7, wherein each binding unit comprises two heavy chains and two light chains, .10 wherein the heavy chains and light chains comprise VII and VL amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ
ID NO: I and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ. ID NO: 25 and SEQ TD
NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ
ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or the VII of any one of SEQ ID NO: 15, SEQ
ID NO:
17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VL
of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22.
(01851 Embodiment 10. The multimeric binding molecule of embodiment 9, wherein each binding unit comprises two heavy chains and two light chains, wherein the heavy chains and light chains comprise VH and VL amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 1 and SEQ ID
NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 13 and SEQ ID NO: .14, or SEQ ID NO:
25 and SEQ ID NO: 26, respectively.
101861 Embodiment 11. The multimeric binding molecule of embodiment 8, wherein the heavy chains and light chains comprise the VH and VI. amino acid sequences SEQ
ID
NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID

NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID
NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO:
30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ
ID NO: 49 and SEQ ID NO: 50, respectively, or the VEI of any one of SEQ ID NO:
15,

- 58 -SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VL of any one of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ
ID
NO: 22.
[01871 Embodiment 12. The multimeric binding molecule of embodiment 11, wherein the heavy chains and light chains comprise the VH and VL amino acid sequences SEQ ID
NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 13 and SEQ
ID NO: 14, or SEQ ID NO: 25 and SEQ ID NO: 26, respectively.
[01881 Embodiment 13. The multimeric binding molecule of any one of embodiments 1 to 10, which is a dimeric binding molecule comprising two bivalent IgA or IgA-like binding units and a J chain or functional fragment or variant thereof, wherein each binding unit comprises two IgA heavy chain constant regions or multimerizing fragments or variants thereof, each comprising an IgA Ca3 domain and an IgA tailpiece domain.
[01891 Embodiment 14. The multimeric binding molecule of embodiment 13, wherein each IgA heavy chain constant region or multimerizing fragment or variant thereof further comprises a Cal domain, a Ca2 domain, an IgA hinge region, or any combination thereof.
[01901 Embodiment 15. The multimeric binding molecule of embodiment 13 or embodiment 14, wherein the IgA heavy chain constant regions or multimerizing fragments thereof are human IgA constant regions.
[01911 Embodiment 16. The multimcric binding molecule of any one of embodiments 13 to 15, wherein each binding unit comprises two IgA heavy chains each comprising a VH
situated amino terminal to the IgA constant region or multimerizing fragment thereof, and two immunoglobulin light chains each comprising a VL situated amino terminal to an immunoglobulin light chain constant region.
[01921 Embodiment 17. The multimeric binding molecule of any one of embodiments 1 to 12, which is a pentameric or a hexameric binding molecule comprising five or six bivalent IgM binding units, respectively, wherein each binding unit comprises two IgM
heavy chain constant regions or multimerizing fragments thereof each associated with a PD-1-binding domain, wherein each IgM heavy chain constant region comprises an IgM
CO and IgM tailpiece domain.
[01931 Embodiment 18. The multimeric binding molecule of embodiment 17, wherein the IgM heavy chain constant regions or fragments or variants thereof each further comprise a Cul domain, a Cp.2 domain, a C1.t3 domain, or any combination thereof.

- 59 -101941 Embodiment 19. The multimeric binding molecule of embodiment 18, wherein the IgM heavy chain constant region is a human IgM constant region.
[01951 Embodiment 20. The multimeric binding molecule of any one of embodiments 17 to 19,wherein each binding unit comprises two IgM heavy chains each comprising a VH
situated amino terminal to the IgM. constant region or fragment thereof, and two immunoglobulin light chains each comprising a VL situated amino terminal to an immunoglobulin light chain constant region.
101961 Embodiment 21. The multimeric binding molecule of any one of embodiments 17 to 20, comprising SEQ ID NO: 35, SEQ ID NO: 36. or a multimerizing fragment thereof.
.10 (01971 Embodiment 22. The multimeric binding molecule of any one of embodiments 17 to 20, wherein the 1gM constant region comprises a substitution relative to a wild-type human IgM constant region at position 310, 311, 313, and/or 315 of SEQ ID NO:
35 or SEQ ID NO: 36.
[01981 Embodiment 23. The multimeric binding molecule of any one of embodiments 17 to 20, wherein the IgM constant region comprises two or more substitutions relative to a wild-type human IgM constant region at positions 46,209, 272, or 440 of SEQ ID
NO: 35 or SEQ ID NO: 36.
[01991 Embodiment 24. The multimeric binding molecule of any one of embodiments 17 to 22 which is pentameric, and further comprises a J-chain or functional fragment or variant thereof.
[02001 Embodiment 25. The multimeric binding molecule of embodiment 24, wherein thel-chain or functional fragment or variant thereof is a variant J-chain comprising one or more single amino acid substitutions, deletions, or insertions relative to a wild-type J-chain that can affect serum half-life of the multimeric binding molecule; and wherein the multimeric binding molecule comprising the variant J-chain exhibits an increased serum half-life upon administration to an animal relative to a reference multimeric binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions, and is administered in the same way to the same animal species.
102011 Embodiment 26. The multimeric binding molecule of embodiment 25, wherein the J-chain or functional fragment thereof comprises an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J-chain (SEQ ID NO: 41).

- 60 -102021 Embodiment 27. The multimeric binding molecule of embodiment 26, wherein the amino acid corresponding to Y102 of SEQ ID NO: 41 is substituted with alanine (A), serine (S), or arginine (R).
[02031 Embodiment 28. The multimeric binding molecule of embodiment 27, wherein the amino acid corresponding to Y102 of SEQ ID NO: 41 is substituted with alanine (A).
[02041 Embodiment 29. The multimeric binding molecule of embodiment 28, wherein the J-chain is a variant human J-chain and comprises the amino acid sequence SEQ ID
NO: 42.
102051 Embodiment 30. The multimeric binding molecule of any one of embodiments 25 .10 to 29, wherein the J-chain or functional fragment thereof comprises an amino acid substitution at the amino acid position corresponding to amino acid N49, amino acid S51, or both N49 and S51 of the mature human J-chain (SEQ ID NO: 41), wherein a single amino acid substitution corresponding to position S51 of SEQ ID NO: 41 is not a threonine (T) substitution.
102061 Embodiment 31. The multimeric binding molecule of embodiment 30, wherein the position corresponding to N49 of SEQ ID NO: 41 is substituted with alanine (A), glycine (0), threonine (T), serime (S) or aspartic acid (D).
(0201 Embodiment 32. The multimeric binding molecule of embodiment 31, wherein thc position corresponding to N49 of SEQ ID NO: 41 or SEQ ID NO: 42 is substituted with alanine (A).
[02081 Embodiment 33. The multimeric binding molecule of any one of embodiments 30 to 32, wherein the position corresponding to 551 of SEQ ID NO: 41 or SEQ ID
NO: 42 is substituted with alanine (A) or glycine (0).
[02091 Embodiment 34. The multimeric binding molecule of embodiment 33, wherein the position corresponding to S51 of SEQ ID NO: 41 or SEQ ID NO: 42 is substituted with alanine (A).
102101 Embodiment 35. The multimeric binding molecule of any one of embodiments 13 to 16 or 24 to 34, wherein the J-chain or functional fragment or variant thereof further comprises a heterologous polypeptide, wherein the hecerologous polypeptide is directly or indirectly fused to the J-chain or functional fragment or variant thereof.
[02111 Embodiment 36. The multimeric binding molecule of embodiment 35, wherein the heterologous polypeptide is fused to the l-chain or fragment thereof via a peptide linker.

- 61 -102121 Embodiment 37. The multimeric binding molecule of embodiment 36, wherein the peptide linker comprises at least 5 amino acids, but no more than 25 amino acids.
[02131 Embodiment 38. The multimeric binding molecule of embodiment 36 or 37, wherein the peptide linker consists of GGGGS (SEQ ID NO: 43), GGGGSGGGGS (SEQ
ID NO: 44), GGGGSGGGGSGGGGS (SEQ ID NO: 45), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 46), Or GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 47).
102141 Embodiment 39. The multimeric binding molecule of any one of embodiments 35 to 38, wherein the heterologous polypeptide is fused to the N-terminus of the 3-chain. or .10 fragment or variant thereof, the C-terminus of the J-chain or fragment or variant thereof, or to both the N-term inns and C-terminus of the J-chain or fragment or variant thereof 102151 Embodiment 40. The multimeric binding molecule of any one of embodiments 35 to 39, wherein the heterologous polypeptide can influence the absorption, distribution, metabolism and/or excretion (ADME) of the multimeric binding molecule.
[02161 Embodiment 41. The multimeric binding molecule of any one of embodiments 35 to 39, wherein the heterologous polypeptide comprises an antigen binding domain.
[02171 Embodiment 42. The multimeric binding molecule of embodiment 41, wherein the antigen binding domain of the heterologous polypeptide is an antibody or antigen-binding fragment thereof.
102181 Embodiment 43. The multimeric binding molecule of embodiment 42, wherein the antigen-binding fragment comprises an Fab fragment, an Fab' fragment, an F(abc)2 fragment, an Fd fragment, an Fv fragment, a single-chain Fv (scFv) fragment, a disulfide-linked FY' (sdFv) fragment, or any combination thereof.
[02191 Embodiment 44. The multimeric binding molecule of embodiment 42 or embodiment 43, wherein the antigen-binding fragment is a scFv fragment.
102201 Embodiment 45. The multimeric binding molecule of any one of embodiments 41 to 44, wherein the antigen binding domain binds ICOS Ligand (ICOSL,G), ICOS
(CD278), Interleukin 6 (II.6), CD28, CD3, CD80, CD86, Tumor Necrosis Factor Alpha (TNFa), or Fibroblast Activation Protein (FAP).
102211 Embodiment 46. A composition comprising the multimeric binding molecule of any one of embodiments 1 to 45.
[02221 Embodiment 47. A polynucleotide comprising a nucleic acid sequence that encodes a polypeptide subunit of the binding molecule of any one of embodiments 1 to 45.

-62 -102231 Embodiment 48. The polynucleotide of embodiment 47, wherein the polypeptide subunit comprises an TOW heavy chain constant region and at least an antibody VH portion of the PD-1-binding domain of the multimeric binding molecule.
[02241 Embodiment 49. The polynucleotide of embodiment 48, wherein the polypeptide subunit comprises a human. IgM constant region or fragment thereof fused to the C-tenninal end of a VI-I comprising:
(02251 (a) HCDR1, HCDR2, and HCDR3 regions comprising the CDRs contained in the VH amino acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7, SEQ ID NO: 9, SEQ ID NO: II, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ
.10 ID NO:
19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
NO: 27, SEQ ID NO 29, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 49, or the CDRs contained in the VH amino acid sequences SEQ ID NO: 1, SEQ ID NO: 3, SEQ
ID
NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:
15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ.
ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ
ID NO: 49 with one or two single amino acid substitutions in one or more of the HCDRs;
or (b) an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ
ID
NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:
19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 49.
[02271 Embodiment 50. The polynucleotide of any one of ein bodiments 47 to 49, wherein the polypeptide subunit comprises a light chain constant region and an antibody VL
portion of the PD-1-binding domain of the multimeric binding molecule.
102281 Embodiment 51. The polynucleotide of embodiment 50, wherein the polypeptide subunit comprises a human. kappa or lambda light chain constant region or fragment thereof fused to the C-terminal end of a VI, comprising:
[02291 (a) LCDR1, LCDR2, and LCDR3 regions comprising the CDRs contained in the VL
amino acid sequences SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ
ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID
NO: 20, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:

32, SEQ ID NO: 34, or SEQ ID NO: 50, or the CDRs contained in the VI, amino acid

-63 -sequences SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:
10, SEQ ID NO: 12, SEQ II) NO: 14, SEQ ED NO: 16, SEQ ID NO: 18, SEQ ID NO:
20, SEQ ID NO: 22, SEQ ID NO: 26, SEQ. ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ
ID NO: 34, or SEQ ID NO: 50 with one or two single amino acid substitutions in one or more of the I.C.DRs; or [0230] (b) an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ
ID
NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:

20, SEQ ID NO: 22, SEQ ID NO: 26, SEQ II) NO: 28, SEQ ID NO: 30, SEQ ID NO:
32, .10 SEQ ID NO: 34, SEQ ID NO: 50.
[0231] Embodiment 52. A composition comprising the polynucleotide of any one of embodiments 47 to 49, and the polynucleotide of any one of embodiments 47, 50, or 51.
[0232] Embodiment 53. The composition of embodiment 52, wherein the polynucleotides are on separate vectors.
102331 Embodiment 54. The composition of embodiment 52, wherein the polynucleotides are on a single vector.
[0234] Embodiment 55. The composition of any one of embodiments 52 to 54, further comprising a polynucleotide comprising a nucleic acid sequence encoding a I
chain, or a functional fragment thereof, or a functional variant thereof 102351 Embodiment 56. The vector of embodiment 54.
[0236] Embodiment 57. The vectors of embodiment 53.
[0237] Embodiment 58. A host cell comprising the poly-nucleotide of any one of embodiments 47 to 51, the composition of any one of embodiments 52 to 55, or the vector or vectors of any one of embodiments 56 or 57, wherein the host cell can express the binding molecule of any one of embodiments 1 to 45, or a subunit thereof.
[0238] Embodiment 59. A method of producing the binding molecule of any one of embodiments I to 44, comprising culturing the host cell of embodiment 58, and recovering the binding molecule.
102391 Embodiment 60. A method for treating an autoimmune disorder, an inflammatory disorder, or a combination thereof in a subject in need of treatment comprising administering to the subject an effective amount of the multimeric binding molecule of any one of embodiments 1 to 45, wherein the multimeric binding molecule exhibits greater

- 64 -potency than an equivalent amount of a monomeric or dimeric binding molecule binding to the same binding partner.
[02401 Embodiment 61. A method for preventing transplantation rejection in a subject, comprising administering to the subject an effective amount of the multimeric binding molecule of any one of embodiments 1 to 45, wherein the multimeric binding molecule exhibits greater potency than an equivalent amount of a monomeric or dimeric binding molecule binding to the same binding partner, and wherein the subject is a transplantation recipient.
102411 Embodiment 62. The method of embodiment 60 or embodiment 61, wherein the .10 subject is human.
[02421 The following examples are offered by way of illustration and not by way of limitation.
Examples Example 1: Materials and Methods 102431 The PathHunter Checkpoint Signaling Assay (Eurofins DiscoverX) is used to determine the relative level of PD-1 signaling induced by antibodies or recombinant proteins. This assay utilizes Jurkat cells, which are modified with an enzyme fragment complementation approach in which portions of the beta-galactosida.se enzyme are split and covalently linked to an intracellular PD-1 signaling domain or the SHP-1 intracellular signaling mediator, which naturally associates with PD-1 during signaling events. In the presence of substrate, PD-1 signaling induces a chemiluminescent signal.

PD-I reporter Jurkat cells are thawed and expanded according to standard cell culture procedures. Cells are seeded at 45 pl., per well of a 96 well plate and antibodies are added 10x final concentration and incubated at 37 degrees Celsius for 30 minutes. PD-I, I+
ligand-presenting cells or additional media are added in 45 jaL volume and are incubated for an additional 2-8 hours. 101.11 of Bioassay Reagent 2 is added, and cells are incubated for 15 minutes at room temperature in the dark. 404 of Bioassay Reagent 2 and cells are incubated for an additional 1 hour in the dark at room temperature. Cherni luminescent signal is measured on a Molecular Devices SpectraMax Paradigm and data are analyzed in GraphPad Prism.

-65 -Example 2: Antibody Generation and Purification Anti-PD-1 IgM #1 and 1i2 and Anti-PD-1 IgG #1 and 42 102451 As exemplary constructs, the VH and VI., regions of two anti-PD-1 antibodies were incorporated into IgM (with an exemplary 3-chain, SEQ ID NO: 41) and IgG
formats according to standard cloning protocols. Anti-PD-1 41 constructs include the VH and VL
amino acid sequences SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and Anti-constructs include the VH and VL amino acid sequences SEQ ID NO: 25 and SEQ ID

NO: 26, respectively. These antibody constructs were expressed and purified according to methods described in W02017196867. The IgM antibodies assembled as pentamers with a i-chain (data not shown).
Example 3: PD-1 Binding by ELISA
102461 An enzyme-linked immunosorbent assay (ELISA) was performed to assess the .15 binding of anti-PD- I. antibodies to human, cynomolgus monkey, or mouse PD- I .
Recombinant proteins containing the extracellular domain of PD-1 together with either an immunoglobulin Fe region or a His tag were used to coat wells of standard 96 well ELISA
plates by overnight incubation at 4 C. Unbound recombinant protein was removed, Pierce SUPERBLOCK'TM T20 was added, and plates were incubated for 10 minutes at room temperature with shaking. Plates were washed with phosphate buffered saline-TWEEN-20 (PBST), and anti-PD- I antibodies were added in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA) and incubated for one hour at room temperature with shaking. Plates were then washed in PBST and incubated with horse radish peroxidase (FERP)-conjugated secondary antibodies in F(ab')2 directed to either human IgG
or lnunan IgM. After one hour, plates were washed again with PBST, signal was developed with SUPERSIGNALTm ELISA Pico substrate, and an ENVISION luminometer (Perk in Elmer) was used to record signal. Data. were analyzed with GRAPHPAD
PRISM .
The results for human PD-1 are shown in FIG. 1. The calculated half maximal effective concentration (EC50) for each antibody and format are shown in Table 2. All antibodies also bound cynomolgus monkey PD-I but did not bind mouse PD-1.

- 66 -Table 2: ELISA EC50 Antibody ______________________ I IgG EC50 (nM) I,gM EC50(nM) #1 1.5 0.087 #2 n.60 0.35 Example 4: PD-I Cell-Based Binding Assay 102471 HEI(.293 cells were modified to overexpress human or mouse PD-1. Cells were expanded in puromycin-containing media to maintain selection, and aliquoted to 96 well V-bottom plates for antibody staining and flow cytornetric analysis. Anti-PD-1 antibodies were added to cells in BD stain buffer with fetal bovine serum (BD
Biosciences, cat. 4 554656) and incubated on ice for 20 minutes. Cells were then washed, and R-phycoerythrin (PE)-labeled anti-human IgG or anti-human IgM secondary-antibodies were then added and incubated for an additional 20 minutes. Data were collected on a Beckman Coulter CYTOFLEX cytometer and analyzed in FLOWJOTM. The results for human PD-1 are shown in FIG. 2. The calculated EC50 for each antibody and format are shown in Table 3. The antibodies did not bind mouse PD- I .
Table 3: Cell Binding ECso Antibody 1gG EC5o (nM) IgM EC5o(nM) #1 59 0.,0 #2 2.9 nia Example 5: PD-1 Signaling Assay 102481 The PATI-11-1UNTERO Checkpoint Signaling Assay (Eurofins DiscoverX) was used to determine the relative level of PD-1 signaling induced by the antibodies.
This assay utilizes an enzyme fragment complementation approach in which portions of the beta-galactosidase enzyme are split and covalently linked to an intracellular PD-1 signaling domain or the SHP-i intracellular signaling mediator. When SHP-1 associates with PD-1 during signaling events, a cherniluminescent signal is generated.

PD-1 reporter Jurkat cells were thawed and expanded according to manufacturer's instructions. Cells were seeded overnight at 40 pi, per well of a 96 well plate and antibodies were added at 10x final concentration and incubated at 37 " C for 1 hour. PD-L1+ ligand-presenting cells or additional media were then added in 40 uL
volume and

- 67 -incubated for an additional 1 hour at room temperature. 10 pl. of Bioassay Reagent 1 (component of Eurofins Discover X cat. # 93-I 104719-001117) was added, and cells were incubated for 15 minutes at room temperature in the dark. 40 pl. of Bioassay Reagent 2 was added, and cells were incubated for an additional 3 hours in the dark at room temperature. Chemiluminescent signal was measured on a PerkinElmer's ENVISION
multilabel plate reader, and GRAPHPAD PRISM was used for data analysis. The results for Antibody #1 and Antibody #2 are shown in FIGS. 3A and 3B, respectively.
The calculated EC50 for each antibody and fomiat are shown in Table 4. IgM-formatted antibodies showed increased potency over IgG-formatted antibodies.
Table 4: PD-1 Signaling ECso Antibody IgG ECso (nM) IgM EC50(nM) 41 65 0.014 #2 ND 0.72 Example 6: PD-1 Signaling Assay 102501 The PD-1 signaling assay described in Example 5 was repeated to determine the relative level of PD-1 signaling induced by crosslinked IgG Antibody #1 and #2. The IgG
antibodies were crosslinked using a "crosslinking antibody" (AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, F(ab1)2 Fragment Specific (Jackson ImmunoResearch, P/N

006-097)). The crosslinking antibody was diluted and added at 10 L per well for a final crosslinking antibody to PD-1 agonist antibody ratio of 1:1.
102511 The resulting PD-1 signaling for Antibody 41 and Antibody 42 compared to the results of Example 5 are shown in FIGS. 4A and 4B, respectively. The calculated EC50 for each antibody compared to the results of Example 5 format are shown in Table 5. IgM-formatted antibodies showed increased potency over IgG-formatted antibodies.
The cross-linked IgG antibodies had an intermediate effect.
Table 5: PD-1 Signaling ECso Antibody IgG ECso (nM) IgG/xlink ECso 1gM
EC50(nM) (nM) #1 65 0.65 0.014 42 ND 4.8 0.72

- 68 -Example 7: Generation and Purification of Additional Antibodies 1.0252]
As additional exemplary constructs, the VII and VL regions of two anti-PD-1 antibodies were incorporated into IOW (with an exemplary J.-chain, SEQ ID NO:
41) and IgG formats according to standard cloning protocols. Anti-PD-I 43 constructs include the VII and VL amino acid sequences SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and Anti-PD-1 #4 constructs include the VH and VL amino acid sequences SEQ ID NO:
3 and SEQ ID NO: 4, respectively. These antibody constructs were expressed and purified according to methods described in W02017196867. The 1gM antibodies assembled as pentamers with a i-chain (data not shown).
Example 8: PD-1 Signaling Assay [02531 The PD-I. signaling assay was conducted according to the method in Examples 5 and 6 with the following modification: the second 1 hr incubation after the addition of additional media was conducted at 4 C for all conditions. The results for Antibody #1-Antibody #4 are shown in FIGS. 5A-5D, respectively. The calculated EC50 for each antibody and format are shown in Table 6. NM-formatted antibodies showed increased potency over IgG-formatted antibodies. The cross-linked IgG antibodies had an intermediate effect.
Table 6: PD-1 Signaling ECso Antibody IgG EC50(nM) IgG/xlink E(.750 IgM EC50 (nM) _______________________________________________ (PM) #1 29 4.4 0.031 #2 5.9 2.1 0.10 #3 0.19 0.19 0.065 0.89 1.7 1.1 Example 9: Antibody Generation and Purification Anti-PD-1 IgG and pentameric IgM #5 102541 As an additional exemplary construct, the VII and VL regions of an anti-antibody was incorporated into IgM (with an exemplar)' 3-chain, SEQ ID NO: 41) and IgG
formats according to standard cloning protocols. Anti-PD-I 45 constructs include the VH
and VL amino acid sequences SEQ ID NO: 49 and SEQ ID NO: 50, respectively.
These

- 69 -antibody constructs were expressed and purified according to methods described in W02017196867. The leM antibodies assembled as pentamers with a I-chain (data not shown).
Anti-PD-1 hexameric IgM (IgHM) #1, 42, #3, and #5 [0255] As exemplary constructs, the VH and VL regions of three anti-PD-1 antibodies were incorporated into IgM without a .1 chain according to standard cloning protocols. The anti-PD-1 #1 Igi-EM construct included the VH and VL amino acid sequences SEQ ID
NO: 13 and SEQ ID NO: 14, respectively, the anti-PD-1 #2 IgHM construct included the VH and VI, amino acid sequences SEQ ID NO: 25 and SEQ ID NO: 26, respectively, the anti-PD-.10 1 43 IgHM construct included the VIT. and VL amino acid sequences SEQ
ID NO: 1 and SEQ ID NO: 2, respectively, the anti-PD-1 45 IgHM constnict included the VH
and VI.
amino acid sequences SEQ ID NO: 49 and SEQ ID NO: 50, respectively. These antibody constructs were expressed an.d purified according to methods described in W02017196867. The IgM antibodies assembled as hexamers without a J-chain (data not shown).
Example 10: PD-1 Signaling Assay [0256] The PD-I signaling assay was conducted according to the method in Example 8 with Antibodies #1413 and 445 in IgG, crosslinked IgG, pentameric IgM, and hexameric IgM
(1gHM) formats. The results for Antibody 41-Antibody #3 and Antibody #5 arc shown in FIGS. 6A-6D, respectively. The calculated FC50 for each antibody and format are shown in Table 7. IgM-formatted antibodies showed increased potency over IgG-formatted antibodies. The cross-linked IgG antibodies had an intermediate effect.
Table 7: PD-1 Signaling EC50 Anti body IgG EC50(nM) IgG/xlink EC50 IgM ECso (nM) IgHM
ECso (nM) (nM) #1 85 1.8 0.057 0.070 #2 22 3.8 0.29 0.40 #3 0.26 0.79 0.13 0.20 45 0.063 0.093 0.030 0.054

- 70 -a Table 8: Anti-PD1 Antibody VH and VI, Sequences e-t eq ISEQ VH or Heavy Chain SEQ VL or Light Chain Reference ID ID
QVQPIQSGAEVKKP GASVKVS CKVSGYS SKIMMSTMIQ DIQMTQS

AP GKGL altar I YT S GYT D YAQKFQGRVTMT EDT ST DTA QQKPGKAPKILI YGASDLP
S GVPS RFS GS GS GTDFT
YMELSSLSEDTAVYYCATGIcP1TNGFNSWGQGTLV1V LT I S S LQPEDEATYY
CQNNYYVGPVS YAFGGGTKVE

QVQLVQ S GS EL K K GASVKVS CKAS TIT FT DY SMEIVRQ

AP GQGLEWMGIIINI ETGEP T YAQG FT GRIVFS LDTSVST EIVLTQS PATLS LS P
GERATL S CTAS S SVS S S YLHW Al AY LQ I S SLICAli17.17AWFCKDYYGTY FYAMDYWGQ GT LV 1QQKPGLAPRLLIYS
TSNTAS GI P DRFSGSGS GTDY

TLTISRLEPEDFAVYYCHQYHRSPLTFGQGTKLEIK
QVTLKESGP GLLQP SQTLS LTC S FSGFS LST S GMGVSWI DIVMTQAALSNPVTL GT

RQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTI SKDTSSN YLNWYLQKPGQS PQLLI
YQMSNLASGVPDRESSSGS Al QVFL KI T =TAM' GTYYCVPKGYYDYGYVMDYWGQGTT GTDET LRI S
RVEAEDVGVYYCAQNLELPLT EGS GT K

QVQLQQSGAELVKPGAS VKLS CKAS GYT FT SYDINWVRQ

RP EQGLEWI GW I FPGDGST KYNEKFKGKATLTTDKS S ST DIVLTQS PS S LSAS L
GERVS LTCRASQEI SGYLSWL Al AYMQFSRLT SEDSAVYFCARGGMRQLGRFVYWGQGTTLT QQKPDGT IKRLI
YAASTLDS GVPKRES GS RSGSDYS

SSLESEDFADYYCLQYASNPYTFGGGTKLEIK
EVQLQQ S GA ELVKP GAS VK LS CT ASG FKVKDT YFHWVKQ

RP DQ GL EWI GRIVSAIC GDT KYAP KLO DKAT T T DT S SN T DIVMTQS PS S LSAS L
GDT I TI TCHASQNINVW LSWY Al AYLQLSRLT SEDTAVYYCVLI YYGFEEGDFWGQGTTLTV QQKP GNVPKIJI, I
YEASNLHT GVP S RFS GS GS GT GFT

SSLQPEDIATYYCQQGQS FP LTFGAGTKLELK
QVQLVQ S GAEVK KP GAS V KVS C KAS Grr YWMEWVRQ DIQMTQS PS S LSASV
'-: Q.\ VGTNVAWY U520180355061 AP GQ GL EWMGEIN PNEGGINYAQKFQ GRVT LTVD. KS I ST QQKPEKAPKS LI
YSASYRYS GVPS RFS GS GS GTDFT Al 1 .1 AYMELS RLRSDDTAVY /CT IDYYDYG GYW GQ GT Days S 12 LT I S S
LQPEDFAT YYCQQYNI YPYT FGQGTKLEIK
EVQLQESGPGLVKPSQTLS LTCTVTGYS I TS DYAVINWI R DVLMTQT PLS LSVTP

t-- Q P PGKKLETRIGY I NY GS T SYNP S LK SRVT S RDT $ KNQ
YLEWYLQKPGQSPKIIIYKVSNRFFGVPDRI SGSGS
FS LKL S SVTAAD TAVYYCARW I G S SAWYFDVWGQGTLVT GT DFT LK I S RVEAED
VGVYFC FQGSEVP FT FGQ GT K
13 VS 1.4 LE I K
eq eq ENVLTQS PFS
LSASVGDRVT I TCRAS S SVI S S YLHW
AH GQGLEW I GNFHPYN DDT KYNEKFKGRVIMTVDKS TTT
YQQKPAKAPKIMIYSTSNIASGVPDRFSGSGS GTSY
VYMEL S SLRSEDTAVYYCARENYGSH GGFVYWGQGTLVT
TLTI SSLQPEDFATYYCGQYNS YP FGGGTKVEI K
N.

a SZQ VIE or Heavy Cha SEQ VL or Light Chain Reference ID ID
EVQLVQ S GAEVK KP GAS .-2.FGYT FTT Y P I EWMPQ

ENVMTQS LSASVGDRVTI

t-t AHGQGLEWI GNFHPYKDDT KINEKFKGRVTMTPDTSTST
YQQKFAKAPKLFI YS TSNLASGVP SRFSGSGS GTDY
VYMEL S SLRSEDTAVYYCARENYG SHGGFVYWGQGTLVT
TLTISSLQFEDFATYYCQQYNSYFLTIGGGTKVEIK
Eml 17 VS 18 EVQLVQ S GAEVK KP GASVKVS CKAFG YT FTT Y P I EWVRQ

ENVLTQS PGTLS LS P GERATL S GRAS S SVI S YLHW
AP GQ GL EWMGN FHPYN DDT KYNEKFK GRVTMT RDT STST
YQQKP GQAPRLWIYS TSN LAS GVP DRFSGSGS GTSY
VYMELSSLRSEDTAVYYCARENYGSHGGFVYWGQGTLVT
TLTISRLEPEDRATYYCQQYN S YPLITGGGTKVEIK

EVQLVQ S GAEVKKF GS SVKVS CKAFGYT FTTYP I EWMRQ

ENVLTQSPFSLSASVGDRVTITCPASSSVISSYLHW
AHGQGLEWI GNFHPYN DDT KYNEKFKGRVTI TVDKSTTT
YQQKPAKAPKIJFIYSTSNLASGVPSRFSGSGS GTDY

VYMELSSLRSEDTAVYYCARENYGSHGGFVYWGQGTLVT

TLTISSLQPEDFATYYCQQYNS YPLTFGGGTKVEIK
VS
EVQLVQ S GAEVK KP GS SVKVS CKIAIGYT FTTYP I EWMRQ

PEGQGLEMI GNFHPYIC DDT KYNEKFKGRWII TADKSTST

23 vs EVQ LVQ S GAEVK KP GS SVKVS CKAS GYT FTT Y P I EVIVRQ

AP GQGLEWMGNFHPYNDDTKYNEKEKGRVTITADKSTST
AYMEL S S LR SEDTAVYYCARENYGSH GGFVYWGQGT LVT

QVQLQESGPGVVKPSGTLS LTCAI SGGS GSGGSIRSTR

YQUPGSSP;IVIYEDNUFSGVPDEFSGSIDSSSN
DKSRNHFSLRLNSVTAADTAVYYCARQDYGDSGDWYFDL
SASLTVSGLKTEDEADYYCQSSDSSAVVFGSGTKLT

EVQLVQSGAEVKKPGASVKVSCKASGYRFTSYGI SWVRQ

P.PG;CiLEWMGW1 SAYNGNTNYAQKLQGRVTMTTDTSTNT
SYELTQPPSVSVSPGQTARITCSCiDALPKQYAYWYQ
AYMELRSLRSDDTATfia5AD S GYVIGQGTQI{PGQAPVMVIYKDTERPSGIPERFSGSSSGTKVTL

Q SADNS ITY RVFGGGT KVTVL
t--QVQ LVQSGAEVK KP GASVRVS CKAS GYTLTSYYTHWVRQ QSALTQPASVSGSPGQS IT

AP GQGLEWMGI NPRGAT n'AQKFQGRVTMT PDT MT WYQH.HPGKAP KLI
IYDVTIN RP SGVSDRFSGSKSGNT
t-t VYMELRNLKSEDTALYYCATAGI YGEDFDYWG LVIV ASLTI
SGLLAEDEGDYYCSSYTIVTNFEVLFGGGTK
t-t 29 SS 30 LTV
as, as, as, .7.1"

erN
a SEQ VH or Heavy Chaiu. _________________ ISEQ VL or Light Chain Reference ID ID
QVQLQES GP GIN STN. CIVSGGS SSGAYYW SV) QSVLTQP PSASGT PGQRVT
I SCSC=SNI GSN SVNW US748880202 eg RQHPGKGLEWIGY1YYNGNTYYNYSLKSLV-iISVa7SKN NQLPGTAP KLLI
IGNNQRPSGVPDRISGSKSGT SA
QFSI: KL S SVTAADTAVYYCARAS DYVNGGYRYMDAFD W S GLQ S ENEMY
CAAND D S LN G PVFGRGT KVT

GAHSEVQLVQS GGGVVQPGRS LRLSCAAS GFT FS SYNCD GVHSDIVMTQSP

RMS'ilVRQAPGKGLENVSAI S GS GGST YYADSVKGRFT I S LAWYQQKPGRAP
KVLIYKAST LES GVP SRFS GS GS G
RDNSKNTLYLQMNSLRkEDTAWYCAKENWGSYFDLWGQ
TDFTLTISSLUEDFATYYCQQSYSTPWTFGQGTKL

QVQLVESGG GVVQPGRS LRLDCKASGIT FSNS Gf4HVIVRQ El VLTQS PATLSLSP
GERATL SCRASQSVSSYLAWY
GKGL EWVAVI WYDG S K RYYAD SVK GRFT SRDNSKNT QQKPGQAPRLLI
YDASNRATGIPARFSGSGSGTDFT

SSLEPEDFAVYYCQQS SNWPRTFGQGTKVEIK
Table 9: Other Sequences in the Disclosure SEQ Nickname Isource) Sequence LP S KDVMQGT DEHVVCKVQHPNGNKEKNVPL PVIAEL P P KVSVFVP PRDG FFGNP RK S KL I
CQAT GF S PRQI
WSWLREGIWGSGVITDQWAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQOASSMCVP
Hunan IgM Constant DUTAIBVEAIPPSEASIFLTKSTKLTCLVTUTTYDSvTISWTRQNGEKVKTHTNISESHPNATFSAVGEA
region IMGT allele SICEDDLINSGERFT=THTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPAD
IGHM*03 (GenBank:
VFVQWMQRGULSPEKYVTSAPMPEPQAPGRYFAHSILTVSEBEWNTGETYTCVVAHEALPNRVTERTVDKS
35 pirl5377681) TGKPTLYNVSINMSDIABTCY

GSASAPTLFPINSCENSPSDTSSVAVGCLAUFLPDSITESWYENNSDISSTRGFPSVLRGGIMATSQVL
LPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQI
QIISTAIREGKQVGSGVITDQVQAEAKESGPIT =ST= I KEE. DWLGQ SMFTCRVDHRGLIFQQNAS SMCVP
Human I gM Constant DQDTAI RVFAI P PS FAS I FLUSTKLTCLVT
DLT TYDSVTISWTPQNGEAVKTHTNISESHPNAT FSAVGEA
region INGT allele SI CEDDVINSGERFTCTVPHTDLP S KQT I
SRPKGVALHRPDVYLLPPAREQLNLRESAT ITCLVTGFSPAD

IGHM* 04 ( Genank : Vr/QWMQRGQPLSPEKTVT SAPMPEPQAPGRYFAHSI
LTVSEE EWNTGET YTCVVAH HAL PNR\rf ERTVDKS
eq sp I P01871.41 ) IGKFT LYNVS DIMS DTAGT CY
eq 37 ASPTSPKVFPLSLCSTUDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQMASGDLYTTSSQLTL
Human IgAl heavy chain EATQCLAGKSVTCHWHYTNPSQDVIVPCPVPSTPETPSPSTPPIPSPSCCHPRLSDHRPALEDILLGSEAN
------------- constant region, e.g., erN
a SEQ Nickname (source) Sequence ID
amino acids 144 to 496 ATLSKSGNTFPPEVELLPPPFEELALNELVTLTCLARGFSPKDVIVRWLQGKELPREKYLTWASRUPSQG
of GenBank AIC59035.1 TTTFAVTSILRVAAEDWKKGDTESCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY
eq ASPTSPKVFPLSLDSTPOGKVVVACLVQGFFPUPLSVTIISESGQNNTARNFPPSOASGDLYTTSSQLTL
Hunan IgA2 heavy chain PATQCPDGKSVTCHVKHYTNSSQDVTVPCRVPPPPPCCHPRLSLHRPAIEDLLLGSEANLTCTLTGLRDASG
constant region, e.g., ATFTWTPSSGKSAVQGPPERDLCGCYSVSSVIPGCANWNHGETFTCTAAHPELKTPLTANITKSGNTFRPE
a, amino acids 1 to 340 of VHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLOGSQELPREKYLTWASRQEPSQGTTTYAVTSILRVA
48 GenBank P01677.4 A E DTh lus. GET F S CMVGH FAL P LAFT
QKT I D RMAGK PT H I NV S VVMAEAD GT C Y
MLLFVLTCLLAVFPAI STKS PI FGPEEVNSVEGNSVS I T CYYP PT SVN RHT RKYWCRQGARGGCI TL
I SSEG
YVSSKYAGRANLTNITENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGIINDTKVYTVDL
GRTVTINCPFKTENAQKRKSLYKIGLYPVLVIDSSGYVNPNYTGRIRLDIOTGQLLFSVVINQLRLSDAG
QYLCQAGDDSNSNKKNADLOiLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCRQSSGENCDVVVNILGK
RAPAFEGRILLNPUKDGSFSVVITGLPIEDAGRYLCGABSDGQWEGSPIQAWLFVNEESTIPRSPTVVX
GVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLT
SRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYVICKWKNIGCQ
ALPSWEGPSKAFVNCDENSRLVSLTLNINTPADEGWYWCGVKQGHEYGETAAVY'VAVEERKAAGSRDVSLA
KADAAPDEKVIDSGFREIENKAIQDPRLFAEEKAVADTRDQADGSRASVDSGSSEEQGGSSRALVSTLVPLG
Precursor Human LVLAVGAVAVGVARAPHRKNVDRVSIRSYRTDISMSDPENSREFGANDNMGASSITQETSLGGKEEFVATTE
39 Secretory Component STTETKEPKKAIRSSKEEAEMAYKDELLUSTVAAEAUGPQEA
40 Precursor Human j Chain MKNHLLFWGVLANFIKAVEWAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLKNRENI
SDFTSPLRTREVYHLSDLCKRCDPTEVELDNQIVTATONICDEDSATETCYTYDRNKCYTAVVPLVYGGET
KMVETALTPDACYPD
41 Mature Human J Chain QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPUTREVYHLSDLCKKC
DPTEVELDNQIVTATONICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD
42 j Chain Y102A nuLation QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPINNPENISDPTSPLRTRFWHLSDLCKKC
DPTEVELDNQIVTATONICDEDSATETCATYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD
43 "5" Peptide linker GGGGS
44 "10" Peptide linker GGGGSGGGGS
45 "15" Peptide linker GGGGSGGGGSGGGGS
46 "20" Peptide linker GGGGSGGGGSGGGGSGGGGS
47 , "25" Peptide Linker GGGGSGGGGSGGGGSGGGGSGGGGS

eq eq N.

Claims (25)

WHAT IS CLAIMED IS:

1 . A multimeric binding molecule comprising two, five, or six bivalent binding units or variants or fragments thereof, wherein each binding unit comprises two IgA or IgM heavy chain constant regions or rnultirnerizing fragnents or variants thereof, each associated with a binding domain, wherein three to twelve of the binding domains are programmed cell death protein 1 (PD-1)-binding domains that specifically and agonistically bind to PD-1, wherein the binding molecule can activate PD-1-mediated signal transduction in a cell at a higher potency than an equivalent amount of a bivalent IgG antibody or fragment thereof comprising two of the same PD-1-binding domains, which also specifically binds to and agonizes PD-1.

2. The multimeric binding molecule of claim 1, wherein the three to twel ve PD-1-binding domains comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
(a) the VH and VL comprise six immunoglobulin complementarity determining regions HCDR I , HCDR2, HCDR3, LCDRI, LCDR2, and LCDR3, wherein the HCDR I , FICDR2, HCDR3, LCDR I , LCDR2, and LCDR3 comprise the CDRs of an antibody comprising the VH and VL of SEQ ID NO: 1 and SEQ ID NO:
2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEX) ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO:
11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively with zero, one, or two single amino acid substitutions in one or more of the HCDRs or LCDRs;
(b) the VH and VL comprise six imrnunoglobulin compleinentarity determining regions HCDR1, HCDR2, HCDR3, LCDR.1, LCDR2, and LCDR3, wherein the HCDR I , HCDR2, HCDR3, LCDR I , LCDR2, and LCDR3 comprise the CDRs of an antibody comprising the VH of any one of SEQ ID NO: 15, SEQ ID NO:
17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VL of any one of SEQ ID NO: .16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ

ID NO: 22 with zero, one, or two single amino acid substitutions in. one or more of the HCDRs or LCDRs;
(c) the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 1 and SEQ ID NO:
2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ
ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO:
11 arid SEQ TD NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 49 and SEQ ID NO: 50, respectively; or (d) the VH comprises an. amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to of any one of SEQ ID NO: 15, SEQ ID
NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 24 and the VL comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to of any one of SEQ ID NO: I 6, SEQ

ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22.

3. The multimeric binding molecule of claim 2, whemin the VH and VL
comprise six immunoglobulin conaplernentanty detemtining relOns HCDR1, HCDR2, HCDR3, LCDR I, LCDR2, and LCDR3, wherein the HCDR1, FICDR2, 1-ICDR3, LCDR1, LCDR2, and LCDR3 cornprise the CDRs of an antibody comprising the VH and V.L
of SEQ
ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 13 and SEQ
D NO: 14, and SEQ ID NO: 25 and SEQ ID NO: 26, respectively with zero, one, or two single amino acid substitutions in one or more of the HCDRs or LCDRs.

4. The multimeric binding molecule of claim 1, which is a dimeric binding molecule cornprising two bivalent IgA or IgA-like binding units and a J chain or functional fragment or variant thereof, wherein each binding unit comprises two IgA heavy chain constant regions or multirnerizing fragments or variants thereof, each cornprisine an IeA Ca3 domain and an IgA tailpiece domain.

5. The multimeric binding molecule of claim 4, wherein:

(a) each IgA heavy chain constant region or rnultirnerizing fragment or variant thereof further comprises a Cal domain, a Ca2 domain, an IgA hinge region, or any combination thereof (b) thc IgA heavy chain constant regions or multimcrizing fragments thereof arc human IgA constant regions; and/or (c) each binding unit comprises two IgA heavy chains each comprising a VH
situated amino terminal to the IgA constant region or multinnerizing fragment thereof, and two immunoglobulin light chains each comprising a VL situated amino terminal to an inununoglobulin light chain constant region.

6. The multirneric binding molecule of claim l , which is a pentameric or a hexameric binding molecule comprising five or six bivalent IgM binding units, respectively;
wherein each binding unit comprises two IgM heavy chain constant regions or multimerizing fragments thereof each associated with a PD-1-binding domain, wherein each IgM
heavy chain constant region comprises an IgM Cn4 and IgM tailpiece domain.

7. The multirneric binding molecule of claim 6, wherein:
(a) the IgM heavy chain constant regions or fragments or variants thereof each further comprise a C1.11 domain, a Cf.i2 domain, a C1.t3 domain, or any combination thereof;
(b) the IgM heavy chain constant region is a human IgM constant region.
each binding unit comprises two IgM heavy chains each comprising a VII
situated amino terminal to the IgM constant region or fragment thereof, and two inununoglobulin light chains each comprising a VL situated amino terminal to an irnrnunoglobulin light chain constant region.

8. The multimeric binding rn.olecule of claim 6, wherein the IgM constant region comprises:
(a) SEQ ID NO: 35, SEQ ID NO: 36, or a multimerizing fragment thereof;
(b) a substitution relative to a wild-type human IgM constant region at position 310, 311, 313, and/or 315 of SEQ ID NO: 35 or SEQ 1D NO: 36; or (c) two or more substitutions relative to a wild-type human IgM constant region at positions 46, 209, 272, or 440 of SEQ ID NO: 35 or SEQ ID NO: 36.

9. The multimcric binding molecule of claim 6, which is pentarneric, and further comprises a J-chain or functional fragincnt or variant thereof.

10. The inultimeric binding rn.olecule of claim 9, wherein the J-chain or functional fragment or variant thereof is a variant J-chain comprising one or more single amino acid substitutions, deletions, or insertions relative to a wild-type J-chain that can affect serum half-life of the multimeric binding molecule; and wherein the multinaeric binding molecule comprising the variant J-chain exhibits an increased serum half-life upon administration to an animal relative to a reference multnneric binding molecule that is identical except for the one or more single amino acid substitutions, deletions, or insertions, and is administered in the same way to the sarne animal species.

11. The multimcric binding molecule of claim 9, wherein thc J-chain or functional fragment thereof comprises:
(a) an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human J-chain (SEQ ID NO: 41);
(b) an amino acid substitution at the amino acid position corresponding to amino acid Y102 of the mature wild-type human .1 -chain (SEQ Ill NO: 41), wherein the amino acid corresponding to Y102 of SEQ ID NO: 41 is substituted with alanine (A);
(c) an arnino acid substitution at the amino acid position corresponding to amino acid Y102 of thc mature wild-typc human J -chain (SEQ ID NO: 41), wherein the j-ehain is a variant human J-chain and comprises the amino acid sequence SEQ ID NO: 42; or (d) an amino acid substitution at the amino acid position corresponding to amino acid N49, arnino acid S51, or both N49 and S51 of thc mature human J-chain (SEQ ID NO: 41), wherein a single amino acid substitution corresponding to position S51 of SEQ ID NO: 41 is not a threonine (T) substitution.

12. The multimeric binding rn.olecule of claim 9, wherein the J-chain or functional fragment or variant thereof further comprises a heterologous polypeptide, wherein the heterologous polypeptide is fused to the 3-chain or functional fragment or variant thereof via a peptide linker comprising at least 5 amino acids, but no more than 25 amino acids.

13. The inultimeric binding rnolecule of claim 12, wherein the heterologous polypeptide is fused to the N-terrninus of the J-chain or fragment or variant thereof, the C-terminus of the J-chain or fragment or variant thereof, or to both the N-terminus and C-terminus of the J-ehain or fragrnent or variant thereof.

14. The multimeric binding molecule of claim 12, wherein the heterologous polypeptide comprises an antigen binding domain, and wherein:
(a) the antigen binding domain of the heterologous polypeptide is an antibody or antigen-binding fragment thereof;
(b) the antigen binding domain of the hetcrologous polypeptide is an antigen-binding fragment of an antibody, and the antigen-binding fragment cornprises an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an Fd fragment, an Fv fragment, a single-chain Fv (scFv) fragment, a disulfide-linked Fv (sdFv) fragment, or any combination thereof (c) the antigen binding domain of the heterologous polypeptide is an antigen-bindin.g fragment of an antibody, and the antigen-bindine fragrnent is a scFv fragment; and/or (d) the antigen binding domain of the heterologous polypeptide is an antigen-binding fragment of an antibody, and thc antigen binding dornain binds 1COS
Ligand (ICOSLG), ICOS (CD278), Interleukin 6 (IL6), CD28, CD3, CD8O, CD86, Tumor Necrosis Factor Alpha (TN:Fa), or Fibroblast Activation Protein (FAP).

15. A composition comprising the multimeric binding molecule of any one of claims 1 to 14.

16. A polynucleotide comprising a nucleic acid sequence that encodes a polypeptide subunit of the binding molecule of any one of claims 1 to 14.

17. The polynucleotide of claim 16, wherein. the polypeptide subunit comprises an 11gM heavy chain constant region and at least an antibody VH portion of the PD-1-binding domain of the multimeric binding molecule; or comprises a light chain constant region and an antibody VL portion of the PD-1-binding domain of the multirneric binding molecule.

18. A composition comprising a polynucleotide comprising a nucleic acid sequence that encodes an 1gM heavy chain constant region and at least an antibody VH
portion of the PD-1-binding domain of the multimeric binding molecule of any one of claims 1 to 1.4, and a polyn.ucleotide cornprising a nucleic acid sequence that encodes a light chain constant region and an antibody VL portion of the PD-1-binding domain of the multimeric binding molecule of any one of claims 1 to 14, wherein the polynucleotides are on separate vectors or on a single vector.

19. The composition of claim 18, further comprising a polynucleotide comprising a nucleic acid sequence encoding a J chain, or a functional fragment thereof, or a functional variant thereof

20. The vector or the vectors of clairn 19.

21. A host cell comprising the composition of claim 18, wherein the host cell can express the binding molecule, or a subunit thereof.

22. A method of producing the binding molecule, comprising culturing the host cell of claim 21, and recovering the binding molecule.

23. A method for treating an autoimmune disorder, an inflammatory disorder,. or a cornbination thereof in a subject in need of treatment comprising administering to the subject an effective amount of the multimeric binding Inolecule of any one of claims 1 to 14, wherein the multimeric binding molecule exhibits greater potency than an equivalent amount of a monomeric or ditneric binding molecule binding to the same binding partner.

24. A method for preventing transplantation rejection in a subject, comprising administering to the subject an effective amount of the multimeric binding molecule of any one of clairns 1 to 14, wherein the multimeric binding molecule exhibits greater potency than an equivalent amount of a monomeric or dimeric binding molecule binding to the same binding partner, and wherein the subject is a transplantation recipient.

25. The rnethod of claim 23, wherein the subject is human.